Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan;
Division of Molecular Marine Biology, Ocean Research Institute, University of Tokyo, Tokyo, Japan
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Abstract |
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Introduction |
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The cichlid species in Lake Tanganyika have been classified into 12 tribes (Poll 1986
). Molecular phylogenetic studies in recent years have resolved some aspects of their phylogeny by exploring various markers, such as allozymes (Nishida 1991, 1997
), the control region of mitochondrial DNA (Kocher et al. 1993
; Sturmbauer and Meyer 1993
; Sturmbauer, Verheyen, and Meyer 1994
), the genes for cytochrome b (Sturmbauer and Meyer 1993
; Sturmbauer, Verheyen, and Meyer 1994
) and subunit 2 of NADH dehydrogenase (ND2; Kocher et al. 1995
) in the mitochondrial genome, and noncoding regions in the nuclear genome (Sültmann et al. 1995
; Mayer, Tichy, and Klein 1998
). Nevertheless, some aspects of their phylogenetic relationships remain unresolved. Moreover, even when some relationships are supported by high bootstrap values in analyses using single-locus markers, such as genes in mitochondrial DNA, the possibility of misinterpretation of phylogeny remains, since the tree obtained is only a gene tree, which might differ from the species tree as a result of incomplete lineage sorting of ancestral polymorphisms during successive rounds of speciation (Nei 1987
; Pamilo and Nei 1988
; Takahata 1989
; Avise 2000
) and/or interspecific hybridization (Avise 2000)
. We cannot ignore this possibility, particularly when we attempt to infer the phylogeny of cichlids in the African Great Lakes, since extensive incomplete lineage sorting has already been reported in studies of cichlid flocks in both Lake Malawi (Moran and Kornfield 1993, 1995
; Parker and Kornfield 1997
; Albertson et al. 1999
; Takahashi et al. 2001
) and Lake Victoria (Nagl et al. 1998
). Thus, the phylogeny of the cichlids in Lake Tanganyika needs to be reevaluated.
In recent years, short interspersed elements (SINEs) have been shown to be powerful phylogenetic markers (Murata et al. 1993, 1996
; Shimamura et al. 1997
; Hamada et al. 1998a
; Takahashi et al. 1998, 2001
; Nikaido, Rooney, and Okada 1999
). These elements are retroposons, and they multiply in genomes via the reverse transcription of RNA that has been transcribed from a parental sequence (Weiner, Deininger, and Efstratiadis 1986
). The random choice of the sites of integration of SINEs and the irreversible nature of the integration at each site are extremely useful in efforts to reconstruct phylogeny, since homoplasy is minimized (Okada 1991
; Cook and Tristem 1997
; Miyamoto 1999
; Shedlock, Milinkovitch, and Okada 2000
; Shedlock and Okada 2000
). The above-mentioned characteristics of retroposons should also be useful for detection of incomplete lineage sorting (Hamada et al. 1998b
; Shedlock, Milinkovitch, and Okada 2000
; Shedlock and Okada 2000
). In the present study, we isolated many members of the AFC family of SINEs, a family that has previously been described in cichlids (Takahashi et al. 1998
; Terai, Takahashi, and Okada 1998
), and used them to investigate the phylogenetic relationships among the ancient lineages of cichlid fish in Lake Tanganyika. We also investigated the existence and extent (if any) of incomplete lineage sorting of ancestral polymorphisms among these fish. This is the first comprehensive report, to our knowledge, to indicate that the analysis of SINE insertions is effective in attempts to discover possible "ancient" incomplete lineage sorting.
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Materials and Methods |
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Results |
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Confirmation of Fixation of Alleles with or Without an AFC SINE
To exclude the possibility that polymorphism was responsible for the presence or absence of a particular SINE in the AFC family in each species, we performed experiments using PCR and additional individuals of three selected species, namely, A. burtoni, E. cyanostictus, and P. microlepis, with primers specific for amplification of SINEs at three selected loci (1265, 259, and 1220; fig. 3
). The results demonstrated the absence of such polymorphism in each case examined. Thus, the loci investigated in the present study were considered fixed, with each allele being either with or without a SINE sequence.
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Discussion |
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The phylogenetic tree shown in figure 2
might provide a basis for the evolution of phenotypes such as breeding behavior during the adaptive radiation of cichlids in Lake Tanganyika. The breeding behavior of cichlids in Lake Tanganyika is characterized by two primary types, mouthbrooding and substrate spawning (Fryer and Iles 1972
; Barlow 1991
). Mouthbrooders incubate their eggs in the buccal cavity, whereas substrate spawners take care of their eggs on a substratum. In Lake Tanganyika, Tilapia, Boulengerochromis, and all of the species in the Lamprologini tribe are substrate spawners, whereas all the other cichlids are mouthbrooders. The tree obtained in the present study clearly indicates that mouthbrooders and substrate spawners are polyphyletic (fig. 2
). This result is consistent with the notion that cichlids have evolved breeding behavior multiple times in different lineages during their adaptive radiation (Barlow 1991
; Sturmbauer and Meyer 1993
).
Possible explanations for the incongruent patterns of insertion of SINE sequences are interspecific hybridization and/or the incomplete lineage sorting of ancestral polymorphism of SINEs. Both of these possibilities should remain in consideration because of the difficulty in distinguishing between them based on the present data. However, if we follow the observation that interspecific hybridization seems rare in the extant faunas of cichlids in East Africa (see discussions in Moran and Kornfield [1993, 1995]
and Parker and Kornfield [1997
]) and postulate that a similar situation also existed when the major lineages in Lake Tanganyika radiated, incomplete lineage sorting may be the more likely explanation. The MVhL clade was strongly supported, as shown in table 3
. Therefore, the length of the internode basal to MVhL (from time X to time Y in fig. 4
) appears to have been sufficient for some SINEs to become fixed in the population. Other SINEs that were amplified more recently within this internode (indicated by the yellow rectangle) might, however, be polymorphic. When a SINE was polymorphic (i.e., presence/absence) at the beginning of the divergence of lineages of the MVhL clade (time Y), this SINE became a possible source of an "incongruent locus." After the divergence of each lineage, an allele with or without a SINE unit at a particular locus might have become fixed stochastically (Pamilo and Nei 1988
). This process might cause incongruence among the gene trees of the investigated loci if one locus became fixed for presence while another was fixed for absence. As a hypothetical example, let us consider a certain individual locus at which a SINE unit was inserted at the time indicated by a red arrowhead in figure 4
. The SINE at this locus had been polymorphic in most lineages during period I (blue rectangle) but was fixed or lost independently in each lineage during period II (green rectangle). Then, the SINE inserted at this locus was shared by species A and D, while it was lost in species B and C, even though species D is more distantly related to species A than to species B and C. Since the process of fixation and loss of a SINE at a locus is stochastic, patterns of presence and absence of SINEs can vary among different loci if multiple speciation events overlap in their time courses or occur during short time intervals. This hypothesis, which assumes the fixation of ancestrally polymorphic alleles, is consistent with the absence of a heterozygote of alleles with and without a sequence of the AFC family in the present matrix (table 3
), which can be detected by coexistence of long and short PCR products from a single individual, and with our failure to detect polymorphisms in extant species (fig. 3
).
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Extensive incomplete lineage sorting has also been reported in cichlids in Lake Malawi. Moran and Kornfield (1993, 1995)
investigated the restriction fragment length polymorphisms (RFLPs) of the mitochondrial DNA of Mbuna (rock-dwelling) species and suggested the existence of ancestral polymorphisms that had been retained by multiple species. Parker and Kornfield (1997)
obtained a similar result when they investigated sequences of the control region of mitochondrial DNA in various species of Mbuna. Albertson et al. (1999)
observed amplified fragment length polymorphisms (AFLPs) in several species of Mbuna in Lake Malawi, and their results supported the persistence of ancestral polymorphisms among extant populations. With regard to non-Mbuna species in Lake Malawi, Takahashi et al. (2001)
reported a transspecies polymorphism of a SINE insertion at a specific locus and suggested that it might be due to incomplete lineage sorting and/or interspecific hybridization among these species. A similar phenomenon has also been reported in cichlids of Lake Victoria. An analysis of DNA sequence variation at four randomly selected loci in the nuclear genome revealed sharing of multiple alleles among nearly all 12 of the tested species, demonstrating that neutral polymorphisms have persisted beyond species boundaries (Nagl et al. 1998
).
The importance of incomplete lineage sorting effects on species phylogeny inference has been suggested from both practical (Nagl et al. 1998
; Streelman et al. 1998
; Albertson et al. 1999
) and theoretical (Nei 1987
; Pamilo and Nei 1988
; Takahata 1989
; Wu 1991
; Tachida and Iizuka 1993
; Lyons-Weiler and Milinkovitch 1997
; Avise 2000
) standpoints. Our results indicated the existence of extensive incomplete lineage sorting at 14 loci among the lineages leading to the tribes Eretmodini, Perissodini, Cyprichromini, Limnochromini, Ectodini, and Lamprologini, as well as the MVHT clade. Therefore, we must be careful when attempting to infer phylogenetic relationships among these lineages, particularly when a single-locus marker, including mitochondrial DNA, is used. Let us consider, for example, the monophyly of the H-lineage (tribes in the MVhL clade excluding the Lamprologini). This monophyly was first proposed from the results of allozyme analysis (Nishida 1991, 1997
) and was supported by studies of sequences of the control region and the gene for cytochrome b of mitochondrial DNA (Sturmbauer and Meyer 1993
; Sturmbauer, Verheyen, and Meyer 1994
) with bootstrap values of 43%89%. The H-lineage was, however, suggested to be polyphyletic from the results of a study of mitochondrial DNA (the gene for subunit 2 of NADH dehydrogenase) by Kocher et al. (1995)
, who placed Tanganicodus irsacae (Eretmodini tribe) in the position of a sister group to the Lamprologini tribe. The present study suggests that monophyly of the H-lineage should be reexamined, since we found that the patterns obtained for five (loci 2, 4, 225, 259, and 1245) of the 14 incongruent loci failed to support monophyly.
Multilocus incongruence might be a general phenomenon that has accompanied rapid speciation during the adaptive radiation of various organisms. Further examples of such a phenomenon might be found among the Galapagos finches, Hawaiian Drosophila, and honey creepers (Mayr 1984
), as well as among fish in certain other lakes (Schliewen, Tautz, and Pääbo 1994
; Strecker et al. 1996
), since these organisms are known to form "species flocks," which are seemingly monophyletic groups of closely related species that coexist in the same areas, as do the cichlids in the African Great Lakes. Moreover, adaptive radiation is not necessarily restricted to organisms that form species flocks, and multilocus incongruence might potentially have been a much more general phenomenon than previously expected. Indeed, it has been proposed that discordance between gene phylogeny and species phylogeny (or between gene phylogenies based on studies of different loci) might be due to incomplete lineage sorting in a wide variety of animals and plants, such as species of mouse (Ohtsuka et al. 1996
), char (Hamada et al. 1998b
), rockfish (Alesandrini and Bernardi 1999
), and Brassica (Tatout et al. 1999
). Another possible example of incomplete lineage sorting has been reported in Felidae (Slattery, Murphy, and O'Brien 2000)
. In this report, two distantly related species in this group were suggested to exclusively share an insertion of a SINE at a specific locus. Although Slattery, Murphy, and O'Brien (2000)
proposed that this result was due to parallel insertion of a SINE sequence at the same site in the genomes of these species, we will need many more data to reject the incomplete lineage sorting hypothesis.
In most studies, including the above examples, evidence for incomplete lineage sorting has come from observation of multiple alleles shared among recently radiated species (transspecies polymorphisms). However, this phenomenon may not be specific to groups that have recently radiated. When radiation of species was ancient enough for the alleles responsible for such transspecies polymorphisms to become fixed, its evidence comes only from discordances among genealogies of different loci. The SINE method appears to be advantageous for detection of such an ancient phenomenon, since, unlike the ordinary methods using sequence data, this method can eliminate the possibility of parallelism, which often cannot be easily distinguished from incomplete lineage sorting as a cause of incongruence among estimated gene trees.
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Acknowledgements |
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Footnotes |
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1 Abbreviations: AFC family, African cichlid family; MVH clade, the clade formed by cichlids of Lakes Malawi and Victoria and Haplochromini; MVhL clade, the clade formed by cichlids of Lakes Malawi and Victoria, the H-lineage, and Lamprologini; MVHT clade, the clade formed by cichlids of Lakes Malawi and Victoria, Haplochromini, and Tropheini; SINE, short interspersed element.
2 Keywords: adaptive radiation
incomplete lineage sorting
Lake Tanganyika
cichlid
retroposon
SINE
3 Address for correspondence and reprints: Norihiro Okada, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan. nokada{at}bio.titech.ac.jp
.
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