Using SINEs to Probe Ancient Explosive Speciation: "Hidden" Radiation of African Cichlids?

Yohey Terai*,1, Kazuhiko Takahashi*,1, Mutsumi Nishida{dagger}, Tetsu Sato{ddagger} and Norihiro Okada*,

* Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan
{dagger} Division of Molecular Marine Biology, Ocean Research Institute, University of Tokyo, Nakano, Tokyo, Japan
{ddagger} Conservation Division, WWF Japan, Minato-ku, Tokyo, Japan


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 
Cichlid fishes of the east African Great Lakes represent a paradigm of adaptive radiation. We conducted a phylogenetic analysis of cichlids including pan-African and west African species by using insertion patterns of short interspersed elements (SINEs) at orthologous loci. The monophyly of the east African cichlids was consistently supported by seven independent insertions of SINE sequences that are uniquely shared by these species. In addition, data from four other loci indicated that the genera Tilapia (pan-African) and Steatocranus (west African) are the closest relatives to east African cichlids. However, relationships among Tilapia, Steatocranus, and the east African clade were ambiguous because of incongruencies among topologies suggested by insertion patterns of SINEs at six other loci. One plausible explanation for this phenomenon is incomplete lineage sorting of alleles containing or missing a SINE insertion at these loci during ancestral speciation. Such incomplete sorting may have taken place earlier than 14 MYA, followed by random and stochastic fixation of the alleles in subsequent lineages. These observations prompted us to consider the possibility that cichlid speciation occurred at an accelerated rate during this period when the African Great Lakes did not exist. The SINE method could be useful for detecting ancient exclusive speciation events that tend to remain hidden during conventional sequence analyses because of accumulated point mutations.

Key Words: exclusive speciation • incomplete lineage sorting • African Great Lakes • cichlid • retroposon • SINE • AFC family


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 
Cichlid flocks of the east African Great Lakes, which consist of Lakes Victoria, Malawi, and Tanganyika, have attracted the interest of evolutionary biologists for more than a century. These species exhibit extraordinary levels of diversity and high species endemicity to each lake as the result of independent explosive adaptive radiation (Fryer and Iles 1972; Greenwood 1984, 1991; Coulter 1991). During the last decade, many molecular phylogenetic studies were conducted to elucidate the phylogenetic relationships among cichlids (for review see Meyer 1993 and Nishida 1997). These studies suggested that cichlids in Lakes Victoria and Malawi are closely related, and that species in both lakes are related to only a portion of the lineages found in Lake Tanganyika, the oldest Great Lake, estimated at 9–12 Myr (Cohen, Soreghan, and Scholz 1993). Until recently, little attention was paid to riverine cichlid species that exhibit pan-African and west African distribution. However, these riverine species constitute an important element of any comprehensive phylogenetic reconstruction of African cichlids; knowledge of their origins is indispensable for the elucidation of founder species in east African lakes and hence for inference of the genetic basis for the adaptive radiation that occurred in those lakes. Recent studies (Sültmann et al. 1995; Zardoya et al. 1996; Streelman and Karl 1997; Mayer, Tichy, and Klein 1998; Streelman et al. 1998) have focused on pan-African and west African species through the use of nuclear markers. Such markers evolve more slowly than mitochondrial DNA, which had been employed extensively in previous phylogenetic analyses of east African cichlids. Recent molecular studies designed to gain inferences from the large framework of cichlid phylogeny again included a combination of analyses of mitochondrial and nuclear genes and morphological characters (Farias, Orti, and Meyer 2000), as well as a reassessment of the mitochondrial cytochrome b gene (Farias et al. 2001). With respect to achieving an accurate African cichlid phylogeny, however, these studies remain preliminary, either because taxon sampling of west African cichlids was insufficient and/or because of poor bootstrap supports for a significant fraction of their relationships.

Another topic of interest is whether the evolution of pan- and west African cichlids was accompanied by incomplete lineage sorting and/or interspecific hybridization, which appears to have occurred multiple times during the evolution of cichlids in the African Great Lakes (Moran and Kornfield 1993, 1995; Parker and Kornfield 1997; Nagl et al. 1998; Van Oppen et al. 2000; Takahashi et al. 2001a, 2001b). If this was indeed the case for the pan- and west African cichlids, then caution must be exercised when attempting to elucidate their phylogeny, given that the genealogies of individual loci may not coincide with their phylogeny (Nei 1987; Pamilo and Nei 1988; Takahata 1989; Avise 2000).

Recently, a series of successful phylogenetic analyses was conducted for African Great Lakes cichlids by investigating insertions of the AFC (rican ichlid) family (Takahashi et al. 1998, 2001a, 2001b) of short interspersed elements (SINEs) into orthologous loci in the nuclear genome (for reviews of the SINE method see Okada 1991; Cook and Tristem 1997; Miyamoto 1999; Shedlock, Milinkovitch, and Okada 2000; Shedlock and Okada 2000). Short interspersed elements multiply via retroposition, and their choice of insertion sites is nearly random (Weiner, Deininger, and Efstratiadis 1986). In the absence of any known specific excision mechanism, SINEs remain at the integration locus indefinitely. These characteristics of SINEs make them advantageous for elucidating phylogenetic relationships; the sharing of a SINE sequence at a specific site can be regarded essentially as a synapomorphy. Short interspersed elements have also been useful for detection of both recent (Hamada et al. 1998; Takahashi et al. 2001a) and ancient (Takahashi et al. 2001b) rapid speciation accompanied by incomplete lineage sorting of alleles. In this article we report use of the SINE method to elucidate the phylogenetic relationships among the ancient cichlid lineages in Africa, as well as testing for the existence of incomplete lineage sorting among these lineages to gain insights into their mode of speciation.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 
Analyses were conducted on 13 species belonging to 11 different genera of pan- and west African cichlids, 7 species belonging to different tribes in Lake Tanganyika, and 2 representative species from Lakes Malawi and Victoria (table 1). Tylochromis polylepis, endemic to Lake Tanganyika, was categorized with pan-African cichlids because this genus exhibits pan-African distribution. Eighteen loci at which AFC SINEs (Takahashi et al. 1998; Terai, Takahashi, and Okada 1998) had been inserted were isolated randomly from genomic libraries of Ophthalmotilapia ventralis (loci 223, 234, 241, 255, and 260), Tropheus moorii (loci 304 and 316), Haplotaxodon microlepis (loci 450 and 454), Tanganicodus irsacae (loci 504 and 509), Tilapia rendalli (loci 904 and 906), Dimidiochromis compressiceps (loci 1223, 1240, 1280, and 1544), and Haplochromis machadoi (locus 1708). Primers to sequences flanking SINEs that were inserted into the above individual loci (table 2) were used in polymerase chain reactions (PCRs) to amplify these loci from the genomes of species listed in table 1. The PCR products were sequenced when confirmation of results was necessary (accession numbers AB100503-AB100592). Construction of the phylogenetic tree and detection of possible incomplete lineage sorting was conducted by hand on the basis of the presence or absence of a SINE sequence at the individual loci listed above (see Results and Discussion). All phylogenetic and molecular biology experiments were performed using standard techniques as described previously (Takahashi et al. 1998).


View this table:
[in this window]
[in a new window]
 
Table 1 Fish Species Analyzed by PCR in this Study.

 

View this table:
[in this window]
[in a new window]
 
Table 2 PCR Primers.

 

    Results and Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 
During the analysis of locus 504, long PCR products were observed among all the endemic species of Lake Tanganyika examined, suggesting the existence of a SINE sequence at this locus (fig. 1A, panel a). Similar results were observed for the representative species from Lakes Malawi and Victoria (table 3). For all other species, either pan- or west African, only short PCR products were observed for locus 504, suggesting the absence of a SINE insertion. The PCR results were confirmed by Southern hybridization using two different types of probes. The first hybridization was performed using a probe specific for sequences of the AFC SINE, and each long PCR product yielded a positive signal (fig. 1A, panel b). In contrast, the shorter PCR products yielded no signal and therefore were considered not to contain a sequence homologous to AFC SINE. The second hybridization was conducted using a probe specific for the flanking sequence of the locus 504 SINE, and all short and long PCR products yielded signals. We confirmed that all PCR products were amplified specifically from locus 504 (fig. 1A, panel c). Although a PCR product of intermediate length was observed for Boulengerochromis microlepis (panel a), the Southern blots confirmed that this locus indeed contained a SINE and that its shorter length was due to the existence of a small deletion near that locus (data not shown). Similarly, locus 241 also showed that species endemic to the east African lakes share a unique AFC SINE sequence (table 3). The above results indicate that the SINE sequences had undergone independent insertion at these two loci in a common ancestor of east African cichlids, and hence suggest their monophyly (hereafter, this clade is referred to as "the east African clade"). The east African clade consists of Trematocara unimaculatum (Trematocarini tribe), Boulengerochromis microlepis (Tilapiini tribe), and Bathybates/Hemibates (Bathybatini tribe), as well as the MVhL lineage, which by an earlier SINE analysis (Takahashi et al. 2001b) is proposed to consist of all the other cichlid tribes in Lake Tanganyika and the endemic cichlids in Lakes Victoria and Malawi. The results for loci 255, 450, 509, 1240, and 1708 were also consistent with monophyly among the above cichlids, although in several species these loci failed to yield a PCR product (table 3, blanks). In all cases these "failed" species were pan- or west African and appear to be old lineages based on the monophyly of the east African clade. Thus, the PCR primers may have failed to anneal to the flanking region of the isolated loci as a result of accumulated mutations.



View larger version (27K):
[in this window]
[in a new window]
 
FIG. 1. Detection of orthologous loci in cichlid species using PCR. The three panels (a–c) in A and B show an agarose gel (top) and two autoradiograms. Panel a, electrophoretic profile of PCR products. Panel b, Southern hybridization of AFC SINE sequences within the PCR products shown in panel a. Panel c, rehybridization of the blot in panel b for detection of a locus-specific sequence flanking the site at which the SINE unit was inserted. Closed and open arrowheads indicate expected mobilities of amplified fragments containing or lacking a SINE insertion, respectively

 

View this table:
[in this window]
[in a new window]
 
Table 3 Presence/Absence of an AFC SINE at Each Locus.

 
A different pattern of SINE insertion was observed at loci 234 and 454 (fig. 1B and table 3), in which PCR products containing SINE sequences were observed among species of the east African clade as well as the pan/west African species Tilapia rendalli, Tilapia buttikoferi, and Steatocranus casuarius. The remaining pan/west African species showed no AFC SINE insertions. These results suggest that the two species of Tilapia and S. casuarius are the closest relatives to the east African clade. The SINE insertion patterns obtained from loci 904 and 1280 were consistent with the above relationship, implying that the two species of Tilapia and S. casuarius are closer to the east African clade than Oreochromis niloticus, Sarotherodon melanotheron, and Tylochromis polylepis. The data for locus 906 also suggested that T. polylepis and Chromidotilapia finleyi may be more distantly related to the east African clade than O. niloticus and S. melanotheron. However, this relationship needs to be confirmed by characterizing positive insertions of other markers at additional loci.

On the basis of the above results, a phylogenetic tree for the cichlids was constructed (fig. 2). Monophyly of the east African clade was suggested by earlier studies using nuclear DNA and/or mitochondrial sequences. However, these studies involved only a limited number of genera from pan- and west Africa (3–6 genera; Sültmann et al. 1995; Zardoya et al. 1996; Streelman and Karl 1997; Mayer, Tichy, and Klein 1998; Streelman et al. 1998; Farias, Orti, and Meyer 2000; Farias et al. 2001). Our data from 11 pan- and west African genera for two loci (241 and 504) as well as from 3 to 10 genera for the other five loci (255, 450, 509, 1240, and 1708; table 3) provide the most comprehensive support for monophyly of the east African clade. The sister group relationship between Tilapia/Steatocranus and the east African clade is consistent with the phylogeny proposed by a sequence analysis of the noncoding nuclear locus DXTU1, in which Oreochromis, Tylochromis, Pelvicachromis, and Hemichromis were included as well (bootstrap value of 75%; Mayer, Tichy, and Klein 1998). That study also suggested (bootstrap value of 66%) that the clade consisting of 7 species of Oreochromis is the sister group to the clade corresponding to our east clade. The results obtained for locus 906 are also consistent with this hypothesis (table 3). Our results support the hypothesis that the ancestral stock, which may formerly have populated west Africa, gave rise to lineages that were the forebears of Anomalochromis, Chromidotilapia, Hemichromis, Nanochromis, Oreochromis, Pelvicachromis, Sarotherodon, Teleogramma, and Tylochromis. The divergence of Tilapia, Steatocranus, and the ancestor of the east African clade then followed this event (see Mayer, Tichy, and Klein 1998, Discussion). Clarification of the detailed relationships among the west African lineages will require further characterization of other loci into which the AFC SINE has inserted.



View larger version (21K):
[in this window]
[in a new window]
 
FIG. 2. A phylogenetic tree based on data from the present study. Numbers in boxes at the nodes and internodes indicate the loci to which SINE sequences were assumed to have retroposed during the indicated period. The PCR products were successfully amplified for all the examined species in table 3 from the loci indicated by boldface numbers, whereas other loci failed to yield a product from one or more of these species. The MVhL lineage, whose monophyly was suggested by an earlier SINE study (Takahashi et al. 2001b), includes cichlids endemic to Lakes Victoria and Malawi, as well as those in Lake Tanganyika, with the exclusion of lineages such as Trematocara (Trematocarini tribe), Boulengerochromis (Tilapiini tribe), Bathybates (Bathybatini tribe), and Hemibates (Bathybatini tribe). The gray circle at the node indicates the period when retention of ancestral polymorphisms (presence or absence of a SINE) was assumed to have occurred at loci 223, 260, 304, 316, 1223, and 1544

 
Although most of the polytomies in our phylogenetic tree were due to the lack of phylogenetically informative loci, those observed among Tilapia, Steatocranus, and the east African clade were due to incongruent patterns of SINE insertion at six loci (223, 260, 304, 316, 1223, and 1544; table 3). At locus 260, an AFC sequence was identified only in T. rendalli and the east African clade (fig. 3, panel a). At locus 316, an AFC SINE was found exclusively in the above cichlid lineages, as wells as in T. buttikoferi (fig. 3, panel b), and a similar result was observed for locus 223 (table 3). The most straightforward interpretation of these results is that T. rendalli represents the closest relative to the east African clade, with T. buttikoferi as the next closest relative. However, other data in our study contradict this interpretation. T. buttikoferi and S. casuarius shared the locus 1544 SINE insertion with the east African clade, but no such insertion was found in T. rendalli (fig. 3, panel c). Locus 1223 showed a similar pattern of AFC SINE insertion. Here, the simplest interpretation is that T. rendalli is not the closest relative to the east African clade. Data for locus 304 further support the premise that S. casuarius, and not T. rendalli, is the closest relative of the east African clade. Taken together, the SINE insertion data for the above six loci suggest conflicting interpretations of which lineage(s) is closest to the east African clade. Importantly, however, none of the insertion patterns were in conflict with the relationships suggested by the other 11 loci in our study, namely, the monophyly of the east African clade and its close relationship with Steatocranus and the two species of Tilapia.



View larger version (25K):
[in this window]
[in a new window]
 
FIG. 3. Incongruent patterns of SINE insertion observed among Steatocranus, two species of Tilapia, and the east African clade. The east African clade is represented by two cichlid species from Lake Tanganyika. Panels a–c show electrophoretic profiles of PCR products amplified from loci 260, 316, and 1544, respectively. Closed and open arrowheads indicate expected mobilities of amplified fragments containing or lacking a SINE insertion, respectively

 
Genealogies of multiple loci that show mutual discordance can generally be explained by either interspecific hybridization or incomplete lineage sorting. In particular, lineage sorting has occurred or is ongoing in cichlids of the African Great Lakes. Ongoing incomplete lineage sorting has been reported for fish in Lake Victoria, based on nuclear sequence analysis (Nagl et al. 1998); for rock-dwelling cichlids (Mbuna) in Lake Malawi, based on mitochondrial haplotypes (Moran and Kornfield 1993, 1995; Parker and Kornfield 1997) and a microsatellite locus (Van Oppen et al. 2000); and for non-Mbuna cichlids in Lake Malawi, based on analysis of AFC SINE insertions (Takahashi et al. 2001a). All of these reports of incomplete lineage sorting were based on observations of trans-species polymorphisms of multiple alleles or haplotypes. By investigating discordant patterns of AFC SINE insertion among 14 loci for the Tanganyikan cichlids, Takahashi et al. (2001b) suggested that there may have been "ancient" incomplete lineage sorting ~5–10 MYA, when the major lineages of this lake diverged, and they proposed that alleles that had been polymorphic during that period have since become fixed stochastically in each lineage (see fig. 4 in Takahashi et al. [2001b] for a scheme showing an example of putative incomplete lineage sorting of SINE insertion). With respect to the present work, the divergence of the ancestors of Tilapia, Steatocranus, and the east African clade may have occurred sufficiently long ago so as to allow such alleles to become fixed, given that their speciation events must be older than the age of the east African clade itself, which includes the major lineages of the Tanganyikan cichlids analyzed in this study (fig. 2). Thus, the present incongruencies among the genealogies of the loci we analyzed are most likely due to "ancient" incomplete lineage sorting, which is more like the phenomenon proposed for cichlids in Lake Tanganyika than the situation observed for these fish in Lakes Malawi and Victoria.

Under what circumstances could the speciation of the ancestral lineages of Tilapia, Steatocranus, and the east African clade have accompanied incomplete lineage sorting? Lineage sorting tends to be incomplete when successive speciation events occur rapidly (Nei 1987; Pamilo and Nei 1988; Takahata 1989). All previous examples of incomplete lineage sorting in cichlids (Moran and Kornfield 1993, 1995; Parker and Kornfield 1997; Nagl et al. 1998; Van Oppen et al. 2000; Takahashi et al. 2001a, 2001b) occurred in the African Great Lakes which are known to exhibit explosive rates of speciation. The speciation events that are the focus of the present study, however, preceded the divergence of the oldest lineages in Lake Tanganyika, which corresponds to the basal node of the east African clade (fig. 2). Thus, it is possible that rapid speciation occurred in riverine ecosystems before Lake Tanganyika was formed. An alternate explanation is that speciation may have taken place in a putative ancient lake that existed prior to the formation of Lake Tanganyika. Phylogenetic analysis of central and east African riverine and lacustrine cichlids using mitochondrial DNA sequences (Salzburger et al. 2002) suggests that some lineages that diverged during the primary lacustrine radiation of Tanganyikan cichlids may have been capable of colonizing surrounding rivers secondarily. If a similar process was involved in the more ancient lacustrine radiation hypothesized in the present study, then the ancestors of Tilapia, Steatocranus, and the east African clade might have been the lineages that fled the ancient lake for the riverine habitat prior to the lake's disappearance. Inclusion of more species into future analyses would be helpful to gain greater insight into how extensively the assumed incomplete lineage sorting or interspecific hybridization event occurred, and thus to test the above hypotheses. Especially, analyses of various lineages of tilapiines may be important, either for this purpose or for reconstruction of a more detailed phylogeny, given that they may consist of diverse lineages (Nagl et al. 2001).

Our work demonstrates that the analysis of SINE insertions is capable of detecting possible ancient incomplete lineage sorting (or interspecific hybridization) that took place even before the divergence of the major cichlid lineages of Lake Tanganyika, during an event that occurred ~14 MYA, as estimated by an earlier allozyme analysis (Nishida 1997). Previous analyses of nuclear and/or mitochondrial markers (Sültmann et al. 1995; Zardoya et al. 1996; Streelman and Karl 1997; Mayer, Tichy, and Klein 1998; Streelman et al. 1998; Farias, Orti, and Meyer 2000, Farias et al. 2001) did not recognize this phenomenon. Given that ancient incomplete lineage sorting (or interspecific hybridization) can be detected only by analyzing genealogic discordance among multiple loci, the failure of several of these studies to recognize the phenomenon can be explained by the fact that only a single locus (or two, in the case of Farias et al. [2000]) was analyzed. Even if multiple loci are analyzed, conventional methods of sequence analysis tend to produce relatively large statistical errors when the subjects are ancient lineages that diverged over a relatively short period (e.g., 30% of the bootstrap support for the sister group relationship between Steatocranus and Tilapia; see fig. 2 in Mayer, Tichy, and Klein 1998). Thus, it may often be difficult to discern such statistical errors from bona fide incomplete lineage sorting in analyses of discordance among topologies obtained from multiple loci. Our data suggest that SINEs could be useful as probes for the analysis of explosive speciation, including events that were "ancient."


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 
The authors thank the Lake Tanganyika Research Group of Kyoto University for the collection and identification of cichlid specimens. This work was supported by a grant-in-aid (to N.O.) for Specially Promoted Research and for Overseas Scientific Surveys from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


    Footnotes
 
Pierre Capy, Associate Editor Back

1 These two authors contributed equally to this work. Back

E-mail: nokada{at}bio.titech.ac.jp. Back


    Literature Cited
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 Literature Cited
 

    Avise, J. C. 2000. Phylogeography: the history and formation of species. Harvard University Press, Cambridge.

    Cohen, A. S., M. G. Soreghan, and C. A. Scholz. 1993. Estimating the age of formation of lakes: an example from Lake Tanganyika, east African rift system. Geology 21:511-514.[CrossRef][ISI]

    Cook, J. M., and M. Tristem. 1997. ‘SINEs of the times'—transposable elements as clade markers for their hosts. Trends Ecol. Evol. 12:295-297.[CrossRef][ISI]

    Coulter, G. W. 1991. The benthic fish community. Pp. 151–199 in G. W. Coulter, ed. Lake Tanganyika and its life. Oxford University Press, New York.

    Farias, I. P., G. Orti, and A. Meyer. 2000. Total evidence: molecules, morphology, and the phylogenetics of cichlid fishes. J. Exp. Zool. 288:76-92.[CrossRef][ISI][Medline]

    Farias, I. P., G. Orti, I. Sampaio, H. Schneider, and A. Meyer. 2001. The cytochrome b gene as a phylogenetic marker: the limits of resolution for analyzing relationships among cichlid fishes. J. Mol. Evol. 53:89-103.[ISI][Medline]

    Fryer, G., and T. D. Iles. 1972. The cichlid fishes of the Great Lakes of Africa: their biology and evolution. Oliver & Boyd, Edinburgh.

    Greenwood, P. H. 1984. African cichlids and evolutionary theories. Pp. 141–154 in A. A. Echelle and I. Kornfield, eds. Evolution of fish species flocks. University of Maine at Orono Press, Orono.

    Greenwood, P. H. 1991. Speciation. Pp. 86–102 in M. H. A. Keenleyside, ed. Cichlid fishes: behaviour, ecology and evolution. Chapman & Hall, London.

    Hamada, M., N. Takasaki, J. D. Reist, A. L. DeCicco, A. Goto, and N. Okada. 1998. Detection of the ongoing sorting of ancestrally polymorphic SINEs toward fixation or loss in populations of two species of char during speciation. Genetics 150:301-311.[Abstract/Free Full Text]

    Mayer, W., H. Tichy, and J. Klein. 1998. Phylogeny of African cichlid fishes as revealed by molecular markers. Heredity 80:702-714.[CrossRef][ISI][Medline]

    Meyer, A. 1993. Phylogenetic relationships and evolutionary processes in east African cichlid fishes. Trends Ecol. Evol. 8:279-284.[ISI]

    Miyamoto, M. M. 1999. Perfect SINEs of evolutionary history? Curr. Biol. 9:R816-R819.[CrossRef][ISI][Medline]

    Moran, P., and I. Kornfield. 1993. Retention of an ancestral polymorphism in the Mbuna species flock (Teleostei: Cichlidae) of Lake Malawi. Mol. Biol. Evol. 10:1015-1029.[Free Full Text]

    Moran, P., and I. Kornfield. 1995. Were population bottlenecks associated with the radiation of the Mbuna species flock (Teleostei: Cichlidae) of Lake Malawi? Mol. Biol. Evol. 12:1085-1093.[Free Full Text]

    Nagl, S., H. Tichy, W. E. Mayer, N. Takahata, and J. Klein. 1998. Persistence of neutral polymorphisms in Lake Victoria cichlid fish. Proc. Natl. Acad. Sci. USA 95:14238-14243.[Abstract/Free Full Text]

    Nagl, S., H. Tichy, W. E. Mayer, I. E. Samonte, B. J. McAndrew, and J. Klein. 2001. Classification and phylogenetic relationships of African Tilapiine fishes inferred from mitochondrial DNA sequences. Mol. Phylogenet. Evol. 20:361-374.[CrossRef][ISI][Medline]

    Nei, M. 1987. Molecular evolutionary genetics. Columbia University Press, New York.

    Nishida, M. 1997. Phylogenetic relationships and evolution of Tanganyikan cichlids: a molecular perspective. Pp. 1–24 in H. Kawanabe, M. Hori, and M. Nagoshi, eds. Fish communities in Lake Tanganyika. Kyoto University Press, Kyoto, Japan.

    Okada, N. 1991. SINEs: short interspersed repeated elements of the eukaryotic genome. Trends Ecol. Evol. 6:358-361.[ISI]

    Pamilo, P., and M. Nei. 1988. Relationships between gene trees and species trees. Mol. Biol. Evol. 5:568-583.[Abstract]

    Parker, A., and I. Kornfield. 1997. Evolution of the mitochondrial DNA control region in the Mbuna (Cichlidae) species flock of Lake Malawi, east Africa. J. Mol. Evol. 45:70-83.[ISI][Medline]

    Poll, M. 1986. Classification des Cichlidae du lac Tanganyika: tribus, genres et espèces. Mém. Acad. Roy. Belg. Cl. Sci. 45:5-163.

    Salzburger, W., A. Meyer, S. Baric, E. Verheyen, and C. Sturmbauer. 2002. Phylogeny of the Lake Tanganyika cichlid species flock and its relationship to the central and east African haplochromine cichlid fish faunas. Syst. Biol. 51:113-135.[CrossRef][Medline]

    Shedlock, A. M., M. C. Milinkovitch, and N. Okada. 2000. SINE evolution, missing data, and the origin of whales. Syst. Biol. 49:808-817.[CrossRef][ISI][Medline]

    Shedlock, A. M., and N. Okada. 2000. SINE insertions: powerful tools for molecular systematics. BioEssays 22:148-160.[CrossRef][ISI][Medline]

    Streelman, J. T., and S. A. Karl. 1997. Reconstructing labroid evolution with single-copy nuclear DNA. Proc. R. Soc. Lond. Ser. B 264:1011-1020.[CrossRef][ISI][Medline]

    Streelman, J. T., R. Zardoya, A. Meyer, and S. A. Karl. 1998. Multilocus phylogeny of cichlid fishes (Pisces: Perciformes): evolutionary comparison of microsatellite and single-copy nuclear loci. Mol. Biol. Evol. 15:798-808.[Free Full Text]

    Sültmann, H., W. E. Mayer, F. Figueroa, H. Tichy, and J. Klein. 1995. Phylogenetic analysis of cichlid fishes using nuclear DNA markers. Mol. Biol. Evol. 12:1033-1047.[Abstract]

    Takahashi, K., Y. Terai, M. Nishida, and N. Okada. 1998. A novel family of short interspersed repetitive elements (SINEs) from cichlids: the patterns of insertion of SINEs at orthologous loci support the proposed monophyly of four major groups of cichlid fishes in Lake Tanganyika. Mol. Biol. Evol. 15:391-407.[Abstract]

    Takahashi, K., M. Nishida, M. Yuma, and N. Okada. 2001a. Retroposition of the AFC family of SINEs (short interspersed repetitive elements) before and during the adaptive radiation of cichlid fishes in Lake Malawi and related inferences about phylogeny. J. Mol. Evol. 53:496-507.[CrossRef][ISI][Medline]

    Takahashi, K., Y. Terai, M. Nishida, and N. Okada. 2001b. Phylogenetic relationships and ancient incomplete lineage sorting among cichlid fishes in Lake Tanganyika as revealed by analysis of the insertion of retroposons. Mol. Biol. Evol. 18:2057-2066.[Abstract/Free Full Text]

    Takahata, N. 1989. Gene genealogy in three related populations: consistency probability between gene and population trees. Genetics 122:957-966.[Abstract/Free Full Text]

    Terai, Y., K. Takahashi, and N. Okada. 1998. SINE cousins: The 3'-end tails of the two oldest and distantly related families of SINEs are descended from the 3' ends of LINEs with the same genealogical origin. Mol. Biol. Evol. 15:1460-1471.[Free Full Text]

    Van Oppen, M. J. H., C. Rico, G. F. Turner, and G. M. Hewitt. 2000. Extensive homoplasy, nonstepwise mutations, and shared ancestral polymorphism at a complex microsatellite locus in Lake Malawi cichlids. Mol. Biol. Evol. 17:489-498.[Abstract/Free Full Text]

    Weiner, A. M., P. L. Deininger, and A. Efstratiadis. 1986. Nonviral retroposons: genes, pseudogenes, and transposable elements generated by the reverse flow of genetic information. Annu. Rev. Biochem. 55:631-661.[CrossRef][ISI][Medline]

    Zardoya, R., D. M. Vollmer, C. Craddock, J. T. Streelman, S. Karl, and A. Meyer. 1996. Evolutionary conservation of microsatellite flanking regions and their use in resolving the phylogeny of cichlid fishes (Pisces: Perciformes). Proc. R. Soc. Lond. Ser. B 263:1589-1598.[ISI][Medline]

Accepted for publication February 9, 2003.