Farias, I.P., G. Ortí, I. Sampaio, H. Schneider, and A. Meyer. 1999. Mitochondrial DNA phylogeny of the family Cichlidae: monophyly and fast molecular evolution of the Neotropical assemblage. Journal of Molecular Evolution, 48: 703-711.
Izeni P. Farias1,2, Guillermo Ortí2, Iracilda Sampaio3, Horacio Schneider3, Axel Meyer4,*
1 Departamento de Biologia, Instituto de Ciências Biológicas, Universidade do Amazonas, 69077-000, Estrada do Contorno 3000, Manaus, Amazonas, Brasil.
2 School of Biological Sciences, 314 Manter Hall University of Nebraska, Lincoln, NE 68588-0118, USA.
3 Departamento de Genética, Centro de Ciências Biológicas, Universidade Federal do Pará, Belém, Pará, Brasil.
4 Department of Biology, University of Konstanz, D-78457 Konstanz, Germany.
* Correspondence to: Axel Meyer, Department of Biology, PO Box M617, University of Konstanz, D-78457 Konstanz, Germany. E-mail: axel.meyer@uni-konstanz.de. Tel: +49 7531 884163, Fax: +49 7531 883018
A mitochondrial DNA (mtDNA) phylogeny of cichlid fishes is presented for the most taxonomically-inclusive data set compiled to date (64 taxa). 16S rDNA data establish with confidence relationships among major lineages of cichlids, with a general pattern congruent with previous morphological studies and less inclusive molecular phylogenies based on nuclear genes. Cichlids from Madagascar and India are the most basal groups of the family Cichlidae, and sister to African-Neotropical cichlids. The cichlid phylogeny suggests drift-vicariance events, consistent with the fragmentation of Gondwana, to explain current biogeographic distributions. Important phylogenetic findings include the placement of the controversial genus Heterochromis basal among African cichlids, the South American genus Retroculus as the most basal taxon of the Neotropical cichlid assemblage, and the close relationship of the Neotropical genera Cichla with Astronotus rather than with the crenicichlines. Based on a large number of South American genera, the Neotropical cichlids are defined as a monophyletic assemblage and shown to harbor significantly higher levels of genetic variation than their African counterparts. Relative rate tests suggest that Neotropical cichlids have experienced accelerated rates of molecular evolution. But these high evolutionary rates were significantly higher among geophagine cichlids.
Key Words: cichlid fishes — phylogeny — mitochondrial DNA — Heterochromis — systematics — DNA substitution rate
Freshwater fishes of the family Cichlidae live throughout Africa, the Neotropics, Madagascar and India. This distribution implies an ancestral Gondwana-wide range dating back at least 130 million years (Ma), and an age of origin ostensibly old in light of the currently available fossil evidence (Lundberg 1993). Monophyly of the family Cichlidae and phylogenetic relationships among the main cichlid lineages were assessed using morphological characters (e.g., Kaufman and Liem 1982; Stiassny 1987; Stiassny 1991) and nuclear DNA markers (Zardoya et al. 1996; Streelman and Karl 1997; Streelman et al. 1998; Fig. 1). The phylogeny of cichlids supports a simple vicariance hypothesis to account for their distribution. In agreement with the pattern of fragmentation of the Gondwana supercontinent (Storey et al. 1995), molecular and morphological studies place the Madagascar and Indian taxa as the most basal groups in the cichlid phylogeny, followed by the African and Neotropical taxa forming putative monophyletic sister groups (Fig. 1). The phylogenetic position of the Congo genus Heterochromis remains unresolved, and disputed and had, before this study, only been studied through morphological data (Oliver 1984; Stiassny 1991; Kullander 1998) - molecular data for it have been missing.
Most molecular phylogenetic investigations to date (e.g., Meyer et al. 1990; Sturmbauer and Meyer, 1992; Moran and Kornfiled 1993; Kocher et al. 1995) have focused on the relationships among East African lacustrine cichlids, which comprise more than 70% of the species in the family (Kullander 1998), and constitute a text book example of adaptive radiations, rapid speciation, and trophic specialization (Futuyma 1997). These studies have shown a remarkably low level of genetic divergence in the face of a very rapid rate of intralacustrine speciation (Meyer et al. 1990). Conversely, the mainly riverine Neotropical cichlids with less than 30% of the species in the family, seem to exhibit larger genetic divergences. Genetic variation at a nuclear locus among African cichlids was about half as that reported for a few Neotropical taxa, suggesting that African cichlids may have a slower rate of molecular evolution (Zardoya et al. 1996). The Neotropical riverine fauna is morphologically diverse, geographically widespread, and seems to conserved ancestral morphological patterns (Kullander 1998), but has been relatively poorly studied with molecular data (Roe et al. 1997; Farias et al. 1998; Martin and Bermingham 1998). Further comparisons of the patterns of phylogenetic divergence (morphological and molecular) and speciation within the major cichlid clades should further our understanding of cichlid evolution and the recent radiation of the East African lineages.
The evolutionary histories of African and New World cichlids seem to differ in fundamental ways. The first review of the entire Neotropical cichlid fauna was published more than 90 years ago by Regan (1906). He separated the genus Cichla from the rest of the Neotropical assemblage (approximately 40 genera), and identified Chaetobranchus as the basal lineage in the radiation of Neotropical cichlids. Since then, the phylogenetic position of Cichla remained controversial and enigmatic (Cichoki 1976; Oliver 1984; Kullander 1986; Kullander 1988; Stiassny 1987; Stiassny 1991), but is of particular importance for the resolution of intrafamilial relationships. Kullander (1998) recently proposed a new phylogeny and classification of the Cichlidae, focusing on the South American assemblage. He placed Retroculus as the most basal genus of an otherwise paraphyletic Neotropical cichlid clade that includes Cichla as well as the African genus Heterochromis. Molecular studies have so far included only few Neotropical genera. Zardoya et al. (1996) included only four (Astronotus, Cichla, Cichlasoma, and Crenicichla) and Sültmann et al. (1995) only three (Cichla, Cichlasoma, and Thorichthys). These few genera were used as representatives of a putatively monophyletic Neotropical clade for the assessment of phylogenetic relationships among the major cichlid lineages. More extensive taxonomic sampling of Neotropical cichlids has been carried out by Roe et al. (1997) and Martin and Bermingham (1998), on Central American heroines, and by Farias et al. (1998), on South American cichlasomines and heroines. In our previous study (Farias et al. 1998), we reported phylogenetic relationships among 24 cichlid taxa, including 19 from South America, one from Central America and four from Africa, based on 550 bp of the mitochondrial 16S rRNA gene. In an attempt to obtain a broader picture of the evolutionary relationships among South American cichlid genera, we present an expanded data set including 47 Neotropical species. Together with representatives of all major African lineages (17 species included) and Madagascar cichlids, the present report is the most complete molecular phylogenetic sample of cichlid taxa to date. Increasing the sampling density in poorly represented clades by the addition of new taxa also intends to break up "long branches" and improve the accuracy of phylogenetic inference (Felsenstein 1978; Hillis 1998).
Sixty-four taxa representing the major groups from the family Cichlidae were included in the present study. Voucher specimens for most South American samples were deposited in museum collections (Appendix 1). Definition of suprageneric groups within the cichlids (e.g., cichlasomines, crenicichlines, geophagines, etc. —see Fig. 2, 3) follows common usage and does not have formal systematic implications (Stiassny 1991). A complete list of suprageneric Neotropical groups is provided by Kullander (http://www.nrm.se/ve/pisces/acara/cichtabl.html.en). The pomacentrid Abudefduf sp. and the embiotocid Cymatogaster aggregata were used as outgroup.
Total DNA was isolated by standard proteinase K, phenol/chloroform extraction (Sambrook et al. 1989). PCR was carried out in 100 μl reaction tubes containing 59 μl H2O, 16 μl (5 mM) dNTP, 10 μl (10X) reaction buffer, 8 μl (25 mM) MgCl2, 1 μl (200 ng) each primer, 5 μl DNA (200 ng/μl) and 2.5U Taq DNA polymerase. The PCR was performed using 25 cycles of 93°C for 1 min, 50°C for 1 min, and 72°C for 2 min. Conserved primers 16Sa-L2510 and 16Sb-H3080 (Palumbi et al. 1991) were used for PCR and sequencing. Some samples were sequenced manually using the Thermo Sequenase cycle sequencing kit (Amersham Life Science) and [a-35S] - labeled dATP. Other samples were sequenced using the BigDye Terminator cycle sequencing ready reaction kit (Applied Biosystems Inc.) on an automated DNA sequencer (Applied Biosystems 310) following manufacturer's instructions. All templates were sequenced completely in both directions. The nucleotide sequence data determined for the present paper were deposited in GenBank (accession numbers: 000000-000000).
DNA sequences were aligned using CLUSTALW 1.5 (Thompson et al. 1994). Settings for CLUSTALW were opening gap cost = 20, extending gap cost = 5. The final alignment comprised 564 positions. Phylogenetic analyses were conducted using PAUP* version 4.0d64 (Swofford 1998). A heuristic search with 50 repetitions using random stepwise additions was performed under maximum parsimony (MP). Non-parametric bootstrapping was based on 100 replicates (each with 10 random addition steps). Gaps were treated as ÒmissingÓ. Minimum evolution (ME) methods (Kidd and Sgaramella-Zonta 1971) were also applied to the data, using maximum likelihood distances based on the HKY + Γ model (Hasegawa et al. 1985; Yang 1993). This model takes into account the transitional bias and among site rate heterogeneity observed for the 16S fragment (Ortí and Meyer 1997). Parameters for this model (transition/transversion rate, proportion of invariable sites, and gamma shape parameter) were estimated by optimizing the data on the MP trees. Comparisons of mean distance measures for each major clade were also based on this model. Constancy of nucleotide substitution rates among Neotropical and African cichlids was tested using relative rate tests based on the two-cluster test of Takezaki et al. (1995), as implemented in the PHYLTEST program (Kumar, 1996). This test examines the equality of the average substitution rate for two monophyletic lineages—in this case African and Neotropical cichlids, using the Madagascar-Indian cichlids as outgroup.
Alternative phylogenetic hypotheses (e.g., Kullander 1998, Stiassny 1991) were tested against the most parsimonious trees obtained with our data using maximum-likelihood ratio tests (Kishino and Hasegawa 1989) and Wilcoxon signed-rank tests (Templeton 1983), as implemented in PAUP* 4.0d64. Most parsimonious trees satisfying topological constraints imposed by the alternative hypotheses were obtained for the 16S data set. These trees were then compared to the most parsimonious "unconstrained" trees.
Of a total of 564 bp considered for the analysis, 274 characters were variable and 205 were phylogenetically informative under parsimony. The mean base composition was 29% A, 22% T, 26% C, and 23% G, confirming a slight over representation of A and C, as observed in the mitochondrial 16S fragment of other fish (Ortí et al. 1996). The estimated transition/transversion rate was 2.48. A bias in favor of transitions over transversions has been found in all genes of the mitochondrial genome examined so far (reviewed in Meyer 1994). The other parameters estimated for the HKY + Γ model were proportion of invariable sites = 0.34 and gamma shape = 0.52. The maximum pairwise divergence value observed among all taxa was 0.20 (uncorrected "p" distance) between Paretroplus polyactis and Teleocichla centrarchus. Most other pairwise comparisons had divergence values p < 0.17. Based on several comparisons of fish taxa at different taxonomic levels, Ortí and Meyer (1997: 93) determined that the 16S fragment reaches a saturation level at approximately p=0.22. Therefore, divergence values among cichlid species found in the present study appear to be below the saturation level for this molecule. All positions and substitution types were thus assigned the same weight in the phylogenetic analyses.
Parsimony analyses resulted in 12 MP trees (length = 1493, consistency index = 0.31, retention index = 0.56) a strict consensus of which is shown in Fig. 2. Differences among the MP trees involve relationships among the Central American cichlids `Cichasoma', Archocentrus, and Petenia, and among the geophagines Mikrogeophagus, Biotodoma, and Geophagus. Minimum evolution resulted in a single tree (score = 2.49; Fig. 3). There is overwhelming agreement between the MP and ME results and previous hypotheses based on morphology and nuclear markers (Fig. 1). All of the above support the monophyly of the Cichlidae, the basal position of the Madagascar/Indian cichlids, and the monophyly of the African and Neotropical cichlids, but disagree on the placement of the controversial genus Heterochromis.
In our analyses, the African genus Heterochromis often grouped with Hemichromis, in both MP and ME trees, defining a monophyletic African clade. Maximum Likelihood ratio tests and Wilcoxon signed-rank tests were applied to test alternative hypotheses regarding the position of Heterochromis. According to Stiassny (1991, her Fig.1.3), Heterochromis forms a group with etroplines but Kullander (1998) placed Heterochromis among the basal Neotropical genera (Fig. 1). Both of these hypotheses were optimized for the 16S data using MP heuristic searches under topological constraints. Constrained searches resulted in 108 equally parsimonious trees satisfying Kullander's hypothesis (length = 1510) and 1792 equally parsimonious trees (length = 1514) satisfying Stiassny's hypothesis. Differences between the best MP tree (Fig. 2, length = 1493) and trees satisfying the alternative hypotheses were not always statistically significant. Templeton tests rejected 81.5% and 95.3% of the trees satisfying Kullander's and Stiassny's hypotheses, respectively. These results suggest the inclusion of Heterochromis, probably basal within a monophyletic African clade. However, maximum likelihood tests did not show significant differences among trees.
The Neotropical cichlids have been divided into the following suprageneric groups, without the implication of a formal classification: heroines, cichlasomines, geophagines, crenicichlines and chaetobranchines (Kullander, 1980, 1983, 1986). Representatives of all these groups were included in the present study, and the above classification is corroborated in part by our molecular data. One of the differences between the ME and MP trees obtained in the present study concerns the monophyly of cichlasomines. While ME supports a cichlasomine clade (bootstrap value = 56%, Fig.3) MP does not (Fig.2). Of the seven cichlasomine genera included, Acaronia, Laetacara and Bujurquina consistently grouped together in both types of analyses, whereas the placement of Nannacara, Cichlasoma and Aequidens varied. The monophyly of heroines was well supported by the 16S data and three major clades were found: (1) Symphysodon (Hoplarchus + Hypselecara); (2) Uaru, Heros and Mesonauta ; and (3)`Cichlasoma', Petenia, Archocentrus and Caquetaia. The relationship of taxa within and among these clades, however, was not well defined. Pterophyllum was identified in the ME and MP trees as the basal genus of the heroine group (Fig. 2 and 3).
Despite low bootstrap values, geophagine monophyly (including the crenicichlines) was supported in all analyses (Fig. 2 and 3). Twelve genera (19 species) were included in the present study. ME and MP trees presented different internal arrangements for the geophagines (Fig. 2 and 3). Bootstrap values of more than 50% were observed only for clades formed by the crenicichlines (Teleocichla + Crenicichla), and acarichthyines (Acarichthys + Guianacara) in the ME and MP trees, respectively. The relationship between Geophagus, Mikrogeophagus, Biotodoma, `G.' brasiliensis, Satanoperca, Apistogramma and Taenicara varied in the different analyses.
Our analyses placed the genus Retroculus as the most basal group in the Neotropical radiation and clearly suggested that Cichla is included in the Neotropical clade. The Cichla + Astronotus arrangement was found in both ME and MP trees, as was found before on a limited set of Neotropical cichlids by nuclear DNA studies (Zardoya et al. 1996). But different arrangements were observed among the major Neotropical lineages (Fig. 2 and 3).
A Cichla-crenicichline clade has been proposed by Stiassny (1991) and more recently by Kullander (1998). However, the position of Crenicichla among Neotropical cichlids remains uncertain (Stiassny 1991, her Fig.1.5), but it clearly does not form a clade with Cichla based on DNA data. In order to test the relationships of Cichla and crenicichlines, we applied statistical comparisons using maximum likelihood ratio and Wilcoxon signed-rank tests. For this analysis we used a simplified data set with only Neotropical taxa and four African genera as outgroup. Unconstrained parsimony analysis resulted in 14 MP trees (length = 967), similar in topology to Fig. 2 (grouping crenicichlines but not Cichla, within geophagines). Constrained searches under Stiassny's hypothesis resulted in 16 MP trees (length > 985) grouping Cichla with crenicichlines outside of geophagines. Templeton tests rejected 88% of trees supporting Stiassny's hypothesis. These results support the grouping of crenicichlines within geophagines, with the exclusion of Cichla. However, maximum likelihood tests did not show significant differences among trees.
Because African and Neotropical cichlids are monophyletic sister groups they are, by definition, of the same absolute age, following their divergence from a most recent common ancestor (MRCA). Path lengths across the ME tree (Fig. 3), from the MRCA to each representative African and Neotropical genus are plotted in Fig. 4. Differences in genetic distances from the MRCA to the tip of each branch represent rate variation among lineages. Higher rates of evolution in Neotropical lineages are suggested by the distribution of these genetic distances. This result also was confirmed by relative rate tests (Takezaki et al. 1995). The Z-statistic (Z = 2.5609) rejected rate constancy (P < 0.05) among African and Neotropical lineages, showing that the average distance between the MRCA and Neotropical cichlids (La = 0.0967) and between African cichlids (Lb = 0.0617) was significantly different (La - Lb = 0.0350 ± 0.0090). We also used relative rate tests only on Neotropical cichlids, to compare evolutionary rates among geophagines, cichlasomines, and heroines, using the other genera as outgroup. We detected significantly higher rates among geophagines (Z = 3.86201, P < 0.05). If geophagines are excluded from the Neotropical clade, the average rate of substitution among Neotropical (La = 0.0828) and African (Lb = 0.0603) cichlids was not significantly different (La - Lb = 0.0225 ± 0.0133, Z = 1.6898). These results suggest that the high evolutionary rates observed among Neotropical cichlids are mostly found among geophagines.
This is the first comprehensive study, based on mtDNA sequence data, to address the phylogenetic relationships among major groups of cichlid fishes. Our results show remarkable agreement with previous hypotheses based on morphology and nuclear DNA sequences (Fig. 1-3). The phylogenetic utility of the 16S mitochondrial gene has been established at several taxonomic levels among vertebrate taxa (e.g., Mindell and Honeycutt, 1990; Hillis and Dixon, 1991; Mindell and Thacker 1996) and freshwater fishes (e.g., Alves-Gomes et al. 1995; Ortí et al. 1996; Murphy and Collier 1997; Ortí and Meyer 1997). In spite of considerable homoplasy, the 16S data set contains reliable phylogenetic signal to establish relationships among major lineages of cichlids. The phylogenetic pattern is completely congruent with the fragmentation of Gondwana and suggests drift-vicariance explanations (Stiassny 1991). Cichlids from Madagascar and India are the most basal group to diverge from ancestral African- Neotropical cichlids which coincides with the drifting of the India-Madagascar subcontinent during the late Jurassic, ca. 150 Ma (Smith et al. 1994). More recently, Neotropical and African cichlids diverged following the opening of the South Atlantic Ocean, close to 65 Ma (Pitman et al. 1993). Similar biogeographic explanations have been proposed for characiform and aplocheiloid fishes, based on the same mitochondrial molecular markers (Murphy and Collier 1997; Ortí and Meyer, 1997).
The present study, based on a large number of South American genera and representatives of all major African lineages, supports the definition of the Neotropical cichlids as a monophyletic assemblage. Within this group some relevant systematic findings are worth mentioning: (i) in contrast to Oliver (1984), Stiassny (1991), and Kullander (1998) (see Fig. 1), this mitochondrial molecular evidence tentatively placed Heterochromis among African cichlids, as sister group to Hemichromis or as the most basal African cichlid. (ii) In agreement with Kullander (1998), our results placed the geophagine-like South American genus Retroculus as the most basal taxon of the Neotropical cichlid assemblage; (iii) the placement of the genus Cichla has been disputed for several years. Our results clearly shown that Cichla belongs to the Neotropical clade, suggesting a close relation with Astronotus, and not with the crenicichlines, as was suggested by Stiassny (1991). This result is also supported by cytochrome b , ATPase 6,8 and two nuclear genes sequences (Meyer, Farias and Ortí, unpublished data). (iv) The crenicichlines are nested within the geophagine group. (v) The present results are in agreement with Stiassny (1991) and Kullander (1996), who suggested that the heroines and cichlasomines are monophyletic sister-taxa, a viewpoint also supported by our previous analysis (Farias et al. 1998). (vi) Our results place Acaronia with the cichlasomines (Fig. 2, 3), in contrast to Stiassny's (1991) arrangement that placed this genus within heroines, and Kullander (1998) who considered Acaronia as the sister group of the cichlasomine-heroine clade. (vii) The Central American species included in the present study (Archocentrus nigrofasciatum, `Cichasoma' citrinellum and Petenia splendida), resulted as expected, closely related to the South American heroines, forming a monophyletic clade with Caquetaia spectabile.
Lacustrine African cichlids have recently undergone one of the most rapid adaptive radiations known for vertebrates (Echele and Kornfield 1984; Greenwood 1984; Meyer et al. 1990; Greenwood 1991; reviewed by Meyer 1993). However, this rapid speciation rate is not correlated with high genetic divergence, since African cichlids are known to exhibit an overall low amount of genetic variation (e.g., Meyer et al. 1990). Recently, Zardoya et al. (1996) found twice as much variation at a nuclear marker (TmoM27) among South American than African cichlids. Our path length analysis and relative rate tests confirm this finding based on nuclear DNA that, although considerably less speciose than their African counterparts, the Neotropical cichlids are extremely more variable at the molecular level. The rate of nucleotide divergence at the 16S mitochondrial fragment in Neotropical cichlids was significantly higher than in the African lineages (Fig. 4). However, the rate acceleration was only significantly higher among geophagines.
More than 65 Ma, ensuing the separation of the Gondwanan fragments, Neotropical and African cichlids followed long and independent evolutionary pathways. Africa and South America have undergone radically different climatic histories since their separation in the Cretaceous. The well-documented paucity of the tropical African flora has been linked to an extensive spread of aridity in that continent following mid-Oligocene global cooling trends (Goldblatt 1993). Biotic factors also may have determined significantly different selective pressures for the two continental ichthyofaunas. For example, notopterids, mormyriforms, knerids, and cypriniforms are freshwater fish groups present in Africa but not in South America. The combination of biotic and abiotic factors may have triggered high extinction rates in Africa, perhaps explaining the currently depauperate characiform fauna in Africa (Lundberg 1993; Ortí and Meyer 1997). Surviving ancestral lineages of African cichlids have recently (< 1 Ma) undergone explosive radiations in the East African lakes, accounting for the high number of extant cichlid species and for their low level of genetic differentiation. The predominantly intralacustrine speciation mode in African cichlids might have been shaped mainly by sexual selection and trophic specialization. In contrast, Kullander (1983) suggested that speciation in riverine cichlids in the Neotropics typically occurred by accumulation of genetic changes in allopatric populations living in different river systems, but not generally associated with great trophic divergence. Neotropical cichlids might have experienced lower rates of extinction and speciation than their African counterparts, preserving primitive characteristics and thereby accumulating higher levels of genetic divergence in some of these lineages.
We thank the many colleagues who provided tissue samples and species identification: J. Alves-Gomes, E. Feldberg, E. Ferreira, M. Garcia, J. Porto, R. Reis, C. Silva, E. Silva, and J. Zuanon. We are also grateful to S. Kullander for comments on earlier versions of the manuscript. This work was supported in part by CAPES, Universidade Federal do Pará, Universidade do Amazonas, and University of Nebraska-Lincoln. IPF is a Ph.D. fellow from CAPES. We also acknowledge support through the National Science Foundation (BSR- 912367, BSR-9119867DEB-9615178), the Max-Planck-Society, and the University of Konstanz to A.M.
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