MBE Advance Access originally published online on December 5, 2005
Molecular Biology and Evolution 2006 23(5):866-873; doi:10.1093/molbev/msj071
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Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005 |
Baleen Whale Phylogeny and a Past Extensive Radiation Event Revealed by SINE Insertion Analysis
* Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan; California Academy of Sciences, San Francisco; Department of Cell Biology, National Institute for Basic Biology, Myodaiji, Okazaki, Japan; and The Institute of Cetacean Research, Tokyo, Japan
E-mail: nokada{at}bio.titech.ac.jp.
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Abstract |
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Baleen whales (suborder Mysticeti) comprise 11 extant species that are classified into four families. Although several phylogenetic hypotheses about these taxa have been proposed, their phylogenetic relationships remain confused. We addressed this problem using short interspersed repetitive element (SINE) insertion data, which now are regarded as almost ideal shared, derived characters at the molecular level. We reconstructed the phylogenetic relationships of baleen whales by characterizing 36 informative SINE loci. One of the intriguing conclusions is that balaenopterids and eschrichtiids radiated very rapidly during a very short evolutionary period. During this period, speciation occurred in balaenopterids and eschrichtiids while newly inserted SINE loci remains polymorphic. Later on, these SINEs were sorted incompletely into each lineage. Thus, there are now inconsistencies among species regarding the presence or absence of a given SINE. This is in sharp contrast to the phylogeny of toothed whales, for which no SINE inconsistencies have been found. Furthermore, we found monophyletic groupings between humpback and fin whales as well as between (sei + Bryde's) whales and blue whales, both of which have not previously been recognized. The comprehensive SINE insertion data, together with the mitochondrial DNA phylogeny that was recently completed (Sasaki, T., M. Nikaido, H. Healy et al. 2005. Mitochondrial phylogenetics and evolution of mysticete whales. Syst. Biol. 56:77–90; Rychel, A. L., T. W. Reeder, and A. Berta. 2004. Phylogeny of mysticete whales based on mitochondrial and nuclear data. Mol. Phylogenet. Evol. 32:892–901), provide a nearly complete picture of the evolutionary history of baleen whales.
Key Words: baleen whales • phylogeny • radiation event • incomplete lineage sorting
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionsSupplementary MaterialAcknowledgementsReferences |
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The order Cetacea (whales) is traditionally classified into two suborders, Odontoceti (toothed whales) and Mysticeti (baleen whales), which comprise more than 80 extant species (Rice 1998
). Their morphologies are highly specialized for adaptation to fully aquatic life, including regression of the hind limbs and loss of hair. The acquisition of an echolocating ability and its accompanying morphological features in toothed whales and the presence of baleen plates instead of teeth in baleen whales are also remarkable. The origin and phylogeny of whales have been of great interest to evolutionary biologists for a long time, but their highly specialized features have made it very difficult to reconstruct the evolutionary history of cetaceans. Recent advances in molecular phylogenetics have begun to shed light on this issue. The cetaceans are now known to be nested within artiodactyls and are closely related to hippopotamuses (Shimamura et al. 1997; Gatesy 1998; Nikaido, Rooney, and Okada 1999). Concerning the problem of toothed whales monophyly or paraphyly, we succeeded in isolating three independent SINE loci, which support a monophyletic origin of toothed whales (Nikaido et al. 2001b), however, except for SINEs, there are few molecular data supporting the traditional hypothesis of toothed whale monophyly (Milinkovitch, Orti, and Meyer 1993; Adachi and Hasegawa 1995; Milinkovitch 1995; Smith et al. 1996). The paraphyly of river dolphins is an important discovery in the systematics of cetaceans (Arnason and Gullberg 1996; Cassens et al. 2000; Hamilton et al. 2001; Nikaido et al. 2001b).
Although the phylogeny of toothed whales as well as the origin of whales has been well studied over the past 10 years, interrelationships among baleen whales have not been explored extensively. Arnason, Gullberg, and Widegren (1993) and Arnason and Gullberg (1994) determined and analyzed mitochondrial DNA sequences of a control region and cytochrome b, respectively, of several baleen whales to resolve their phylogenetic relationships. These two analyses yielded the following three consistent conclusions. Balaenidae (bowhead and right whales) are monophyletic at the basal of mysticetes. Neobalaenidae (pygmy right whale) diverged next, followed by minke whales and by sei and Bryde's whales, which are monophyletic. The phylogenetic relationships of species among Balaenopteridae (humpback, fin, sei/Bryde's, and minke whales) and Eschrichtiidae (gray whale) are not, however, well resolved. For example, in the tree reconstructed by the analysis of the control region (Arnason, Gullberg, and Widegren 1993), fin and blue whales are monophyletic, whereas in the phylogenetic tree of cytochrome b (Arnason and Gullberg 1994), humpback and blue whales are monophyletic. Both trees nested gray whales within balaenopterids, showing paraphyly of Balaenopteridae, although the exact phylogenetic position of the gray whale is different between the two studies. Indeed, each topology suggested by different genes is not supported by enough bootstrap values, implying the difficulty of clarifying the relationships among Balaenopteridae and Eschrichtiidae.
Recently, Sasaki et al. (2005) determined the complete mitochondrial genome sequence of 10 extant baleen whales and constructed a phylogenetic tree of Mysticeti by adding the data of blue and fin whales from the GenBank database. Those results showed the monophyly of the Eschrichtiidae + Balaenopteridae and the presence of four principal lineages among the Eschrichtiidae + Balaenopteridae—lineage I (the two minke whales), lineage II (the fin and humpback whales), lineage III (the sei, Bryde's, and blue whales), and lineage IV (the gray whale)—but the data failed to resolve the relationships among these four lineages. The failure in resolving the relationships among these four major lineages is present also in the case of Rychel, Reeder, and Berta (2004), where the partial mitochondrial and nuclear DNA sequences were analyzed.
In our present study, we applied a short interspersed repetitive element (SINE) insertion analysis (Okada 1991a, 1991b; Okada, Shedlock, and Nikaido 2004) to clarify the evolutionary history of mysticetes, with special reference to that of balaenopterids and eschrichtiids.
SINEs are retroposons that are amplified via cDNA intermediates and are reintegrated into the host genome by retroposition (Rogers 1985; Weiner, Deininger, and Efstratiadis 1986; Okada 1991a, 1991b; Kazazian 2000). The integration of a SINE is irreversible, and the probability of independent insertions of a SINE at the same genomic position in different lineages is infinitely small. The polarity of a SINE insertion is fixed from the absence to the presence. These attributes imply that SINEs represent ideal, homoplasy-free markers for inferring phylogenetic relationships among organisms (Shedlock and Okada 2000; Okada, Shedlock, and Nikaido 2004). By applying the SINE method, we recently clarified the phylogeny of cetaceans and their relatives (Shimamura et al. 1997; Nikaido, Rooney, and Okada 1999, Nikaido et al. 2001b).
In the present study, we used the SINE method to reconstruct a phylogenetic tree of 11 extant baleen whale species. We identify an extensive radiation event during speciation of the common ancestors of balaenopterids and eschrichtiids. We also establish the phylogenetic relationships of baleen whales. We show the power of the SINE method not only for inferring the phylogeny of baleen whales but also for detecting an ancient incomplete lineage sorting, which implies a past radiation event during baleen whale evolution.
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Materials and Methods |
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Fourteen cetaceans species (11 mysticetes and 3 odontocetes; all of the DNA samples used in this study are described in supplemental table 1, Supplementary Material online) were examined in this study, using hippopotamus as an outgroup (Nikaido, Rooney, and Okada 1999
). Their DNAs were extracted using phenol/chloroform and then precipitated by ethanol according to Blin and Stafford (1976). The genomic libraries were constructed for all baleen whale species except for pygmy right whale and bowhead whale, for which we had insufficient DNA. We screened these libraries using the CHR-2 SINE (especially the cetacean specific deletion [CD] subfamily) sequence as a probe, taking into consideration the timing of amplification of SINE subfamilies and the phylogeny of whales (Nikaido et al. 2001a, 2001b). Positively hybridized clones were sequenced and then primer sets were designed. All primer sets used in this study are shown in supplemental table 2 (Supplementary Material online). Polymerase chain reaction (PCR) was performed with these primer sets for each SINE locus using cetacean and hippopotamus DNAs as templates. The PCR products were then separated by electrophoresis; larger PCR products indicated the presence of the SINE. To confirm the presence or absence of SINE and the precise site of SINE insertions at particular loci, we roughly sequenced a short surrounding region of each insertion site for almost all bands in the gels (supplemental figures 3 and 5, Supplementary Material online). For phylogenetic analysis, the SINE insertion data were compiled into the data matrix, in which the absence of a SINE at a particular locus was coded as 0, and the presence of a SINE at that same locus was coded as 1 (supplemental table 3, Supplementary Material online). In cases where a PCR band was not visible, the character state was coded as missing (denoted by ?). The resultant data matrics were applied to PAUP* (ver. 4. 0b10; Swofford 1998) for reconstruction of a strict consensus parsimony tree. The analysis was carried out under "IRREV.UP" option, regarding "0"as the ancestral state. Because the polarity of a SINE insertion is fixed, there is no need to root the resultant phylogeny. |
Results and Discussion |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionsSupplementary MaterialAcknowledgementsReferences |
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Phylogenetic Relationships of Baleen Whales
We prepared genomic libraries of nine species of baleen whales and characterized 36 SINE loci, all of which are informative in terms of phylogenetics of cetaceans (all PCR patterns of the 36 loci are shown in supplemental figure 1, Supplementary Material online). It should be noted that SINEs were screened from genomic libraries of almost all species examined, thereby eliminating ascertainment bias that might be derived from choosing a single species as the source of SINE loci for the analysis. We confirmed the fixation of SINE insertions by examining several (3–10) individuals for each species using SINE flanking PCR experiments (we did not confirm right, bowhead, pygmy right, and gray whales because we did not have enough individuals for these species). No within-species polymorphisms, with respect to the presence or absence of a SINE, were detected for the loci characterized here (data not shown).
Figure 1 shows the PCR patterns of 12 SINE loci, which represent each clade of A–I and inconsistent loci J (described later). Clade A represents the monophyly of the order Cetacea, which is supported by five newly isolated loci (figs. 1 and 2a). Clade B represents the monophyly of the suborder Mysticeti (baleen whales), which is supported by five independent loci (figs. 1 and 2a). Because we did not identify any loci that support the sister relationship of baleen whales and sperm whales, we could not validate the monophyly of these two groups, which have been once proposed (e.g., Milinkovitch, Orti, and Meyer 1993; Milinkovitch 1995).
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We elucidated the order in which baleen whales diverged. The right whales and bowhead whales, which are monophyletic (figs. 1 and 2a; clade I), diverged first among baleen whales as shown by five SINE loci (figs. 1 and 2a; clade C). The pygmy right whale diverged next as shown by five SINE loci (figs. 1 and 2a; clade D). Clade C represents the monophyly of Neobalaenidae, Eschrichtiidae, and Balaenopteridae, and clade D represents the monophyly of Eschrichtiidae and Balaenopteridae. Because clades C and D were supported by five independent SINE loci that were isolated and characterized from genomic libraries of more than two species, these two clades remain robust. These results are consistent with previous molecular studies (Arnason, Gretarsdottir, and Widegren 1992
; Adegoke, Arnason, and Widegren 1993; Arnason and Gullberg 1994) and are supported by the high bootstrap values calculated in our recent complete mitochondrial DNA study (fig. 2b, Sasaki et al. 2005). They are also consistent with morphological studies (e.g., Barnes and McLeod 1984; Mead and Brownell 1993).
We further found the presence of the four major lineages that radiated from a common ancestor of Eschrichtiidae and Balaenopteridae. One lineage (I) is a monophyletic group of common and Antarctic minke whales, which is supported by three loci (figs. 1 and 2a; clade E). The second lineage (II) consists of humpback and fin whales, which is supported by three loci (figs. 1 and 2a; clade F). The discovery of this clade is of special interest from the viewpoint of baleen whale systematics. The humpback whale is classified in the genus Megaptera, members of which are morphologically distinct from those of the genus Balaenoptera, which includes fin, blue, sei, Bryde's, and minke whales. The present study supports previous molecular evidence (Adegoke, Arnason, and Widegren 1993; Arnason and Gullberg 1994) that the genus Megaptera is nested within the genus Balaenoptera. Regarding the sister species of humpback whales, however, results of previous molecular studies are confusing. The findings of Baker et al. (1993) recovered the monophyly of humpback and fin whales by the analysis of partial sequences of the mitochondrial control region of baleen whales, but these investigators did not stress this issue. Furthermore, the analysis of the complete sequence of the mitochondrial control region by Arnason, Gullberg, and Widegren (1993) showed very weak (below 50% bootstrap percentage) grouping of fin and blue whales. Thus, no obvious conclusion concerning the relationships of humpback and fin whales was proposed from the analyses of mitochondrial control region. Next, the sister relationship between humpback and blue whales is supported by the analysis of mitochondrial cytochrome b (Arnason and Gullberg 1994). The analysis of nuclear DNA sequences of intron fragments from the actin gene by Cipriano and Palumbi (1999) connected humpback and fin whales as sibling species, although only four species were included in the analysis. The analysis of common cetacean nuclear satellite DNA by Arnason, Gretarsdottir, and Widegren (1992) suggests a blue and fin whale clade. As described above, the sister species of humpback whales has not been clarified. Taking the facts described above, the grouping between humpback and fin whales is quite striking, even for molecular phylogeneticists, morphologists, and paleontologists, because humpback whales possess distinct morphological characters compared to other balaenopterids (e.g., enlarged flippers). Large-scale changes in the developmental systems of humpback whales in their own lineage might make their features distinct and may have created an inconsistency between morphological and molecular phylogenies, resulting in the paraphyly of Balaenoptera.
Lineage III is a monophyletic group of sei, Bryde's, and blue whales that is supported by two loci (figs. 1 and 2a; clade G). Within this clade, the monophyly of sei and Bryde's whales is supported by three loci (figs. 1 and 2a; clade H). Although the monophyly of sei and Bryde's whales has been supported by several lines of evidence for these more than 10 years (Arnason, Gullberg, and Widegren 1993; Arnason and Gullberg 1994, 1996), the sister relationship of blue whales to (sei + Bryde's) whales has been recovered by very recent two molecular studies (Rychel, Reeder, and Berta 2004, Sasaki et al. 2005). Rychel, Reeder, and Berta (2004) compared partial mitochondrial and nuclear DNA sequences, and Sasaki et al. (2005) compared complete mitochondrial genomes resulted in the same topology. The clade G supported by the present SINE data is consistent with these two analyses. Lineage IV is a gray whale lineage (fig. 2a).
Although the monophyly of each of the lineages I–IV is strongly supported by multiple SINE insertion data, the order in which they branched remains unresolved (see below). Figure 2a is a phylogenetic tree with multifurcation of these four lineages (hereafter referred to as a consensus tree).
Characterization of Inconsistent SINE Loci Suggests a Radiation Event from a Common Ancestor of Balaenopterids and Eschrichtiids
During our extensive analysis of SINE insertions in the genomes of baleen whales, we characterized three interesting SINE loci, two of which (BRY28, Sei23) are inconsistent with the consensus tree shown in figure 2a, and all of which are inconsistent with one another. For example, the locus IWA31 (figs. 1 and 2c) indicates monophyly of lineages III and IV (sei, Bryde's, blue, and gray whales), whereas locus BRY28 indicates monophyly of lineage III (sei, Bryde's, and blue whales) and fin whales. Thus, these loci contradict each other, and locus BRY28 violates the monophyly of fin and humpback whales. Furthermore, locus Sei23 indicates the monophyly of lineage I (common and Antarctic minke whales) and sei/Bryde's whales, thus repudiating the clade G. Locus Sei23 is also inconsistent with BRY28 and IWA31, although these two are consistent with clade G. The presence or absence of the SINE for each locus was confirmed by sequencing the PCR bands, and the partial alignments of these loci are shown in supplemental figure 3 (Supplementary Material online). Such inconsistencies were also reported in the recent study by Rychel, Reeder, and Berta (2004) who analyzed baleen whale phylogeny using mitochondrial and nuclear DNA data, where the topologies for each gene was inconsistent. These phylogenetic inconsistencies were detected among the balaenopterids and eschrichtiids, that are very similar to those obtained from our SINE analysis.
These inconsistent loci prompted us to construct the most parsimonious tree using the PAUP* program (Swofford 1998). Supplemental table 3 (Supplementary Material online) shows the data matrix for the presence or absence of a SINE at all 36 loci. Because of the three inconsistent loci, the bootstrap value for connecting the two clades of lineages III and IV was low (59%; see supplemental figure 2, Supplementary Material online), making it reasonable for these two clades to be positioned as multifurcations (fig. 2a; J), which again recovered the consensus SINE tree.
The SINE Tree Versus the Complete Mitochondrial DNA Tree
A complete mitochondrial genome analysis of almost all baleen whale species was recently completed (Sasaki et al. 2005). The resulting proposed phylogenetic tree (fig. 2b) is essentially consistent with the SINE tree generated from the present data (fig. 2a), in which several clades, such as the monophyly of humpback and fin whales (lineage II) and monophyly of Bryde's/sei and blue whales (lineage III), are supported by high bootstrap probabilities. However, the bootstrap probabilities of each of the nodes that give the branching order of lineages I–IV are quite low. It is possible that these low bootstrap values might be a result of rapid, successive divergence events of these four lineages during baleen whale evolution. In that respect, the mitochondrial DNA tree is largely consistent with the SINE consensus tree. Furthermore, a comprehensive analysis of the cetartiodactyl mitogenomic data by Arnason, Gullberg, and Janke (2004) showed a quite similar conclusion in that the phylogenetic relationships of Eschrictiidae and Balaenopteridae are not clear. Their phylogenetic tree showed that the branches joining each baleen whale tended to be short, implying that these groups underwent a rapid split.
Ancestral Polymorphisms, Incomplete Lineage Sorting, and Radiation
As described in Introduction, SINE inserts can provide an excellent record of biological history that is largely free from character reversals and parallel evolution. Namely, the possibility that a SINE is independently inserted in the same genomic locus in different lineages is extremely small (Shedlock and Okada 2000). Accordingly, the history of each SINE at a particular genomic locus represents a gene tree of that locus. Even though there are several individual SINE loci that are inconsistent with one another, each SINE should represent its respective true gene tree. Such inconsistencies might have been generated by ancestral SINE polymorphisms followed by incomplete lineage sorting, which have been frequently observed in cases of rapid successive speciation (Shedlock, Takahashi, and Okada 2004). Supplemental figure 4 (Supplementary Material online) illustrates how an ancestral polymorphism in a common ancestor of the radiated species, followed by incomplete lineage sorting, creates inconsistent SINE patterns during evolution (see Nei and Kumar 2000).
In previous analyses of the origin of whales and the phylogeny of toothed whales using SINEs, inconsistent SINE loci were not detected (Shimamura et al. 1997; Nikaido, Rooney, and Okada 1999; Nikaido et al. 2001b). It is only at the clades F and G that we found inconsistencies in cetacean phylogenetics, suggesting that, at the time of divergence of Balaenopteridae and Eschrictiidae, baleen whales experienced a radiation event. It should be noted that we found no inconsistent loci for clades A, B, C, D, E, and H, supporting strongly each of these monophyletic groupings.
One noteworthy conclusion of the present study is that lineages I–IV of balaenopterids and eschrichtiids radiated very rapidly over a short evolutionary period. This event is highlighted by the gradated box on the phylogenetic tree of baleen whales (fig. 2a; J). This possible radiation was deduced by inconsistent SINE patterns for three independent loci (fig. 1), which might have resulted from ancient incomplete lineage sorting of the SINE polymorphisms that were retained among the common ancestors of extant balaenopterids and eschrichtiids after the divergence of pygmy right whales (a hypothetical ancestor of balaenopterids and eschrichtiids; J). This suggests that the period around J is short. Considering that the effective population size is also one of the most critical factors affecting incomplete lineage sorting (Nei 1987; Pamilo and Nei 1988; Takahata 1989; Shedlock, Takahashi, and Okada 2004), our data indicate that the population size of ancestral whales around J might have been relatively large or might have expanded during successive radiation events of baleen whales. In the previous study of Sasaki et al. (2005), the period of divergence for J was calculated as 
19 million years before present (MYBP). They also suggested that almost all (10 of 12) major extant baleen whale lineages arose during this period, between the early Miocene (23 MYBP) and Middle Miocene (10 MYBP). There is a rich amount of fossil data of the stem group for Balaenopteridae, implying the prosperity of this lineage (Sasaki et al. 2005). Taking the SINE data and the molecular timescale of mitochondrial sequences into account, the baleen whale ancestors have experienced rapid radiation events, especially around 19 MYBP, which are represented by the discovery of extinct stem-balaenopterid groups (Parabalaenoptera, Aglaocetus, and Cophocetus, Sasaki et al. 2005). The data described above have prompted us to explore the oceanic and geographic evidence of past environmental changes that might have occurred around 19 MYBP, an analysis that will help to elucidate the true history of cetacean evolution.
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Conclusions |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionsSupplementary MaterialAcknowledgementsReferences |
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We propose that the isolation of multiple incongruent SINE loci indicates the presence of a radiation event in a common ancestor of Balaenopteridae and Eschrictiidae. During this radiation, successive speciation occurred (Shedlock, Takahashi, and Okada 2004
). Polymorphic SINEs in the past could not have become fixed during such a short period and were incompletely sorted into lineages, resulting in incongruence of SINEs among different lineages. Predicting the presence of a radiation event based on phylogenetic analysis by DNA sequence comparison is difficult because radiation causes low bootstrap values, which are not necessarily caused by radiation. Taking this issue into account, SINE insertion data may form the basis for an ideal method for detecting lineage sorting effects because it enables us to analyze large data sets of multiple nuclear loci independently (Hillis 1999; Miyamoto 1999; Shedlock, Milinkovitch, and Okada 2000) and provides strong reliability when inferring a gene tree (Shedlock, Takahashi, and Okada 2004). The validity of the SINE method for tracking ancient radiation events has been well demonstrated and is yet another benefit of this useful phylogenetic marker. Inconsistent SINE patterns have also been detected among the genomes of African cichlid fishes in Lake Tanganyika (Takahashi et al. 2001) and in ancestral river populations of cichlids (Terai et al. 2003). We predict that future SINE analyses will detect such inconsistencies in many other lineages in which radiation events might have occurred, such as turtles, ruminants, felids, and primates. |
Supplementary Material |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionsSupplementary MaterialAcknowledgementsReferences |
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Supplemental figures 1–5 and tables 1–3 are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).
SUPPLEMENTAL FIG. 1.—PCR patterns of 36 SINE loci. Electrophoretic gel patterns of PCR products for all 36 SINE loci characterized in this study. Bands containing a SINE are indicated by filled arrowheads, whereas open arrowheads indicate the absence of a SINE. Bands containing additional and independent SINE insertions are indicated by striped arrowheads. Loci are assigned alphabetically from A to J according to the clade on the phylogenetic tree shown in figure 2. The name of each locus is based on the whale species from which the genomic library was constructed (BLU and Sir, blue; BRY, Bryde's; Fin and Nag, Fin; GRY, gray; Hump, humpback; Mnk and Ac, Antarctic minke; NM, common minke; Sei and IWA, sei; sNR, NR and SEM, right). The species are numbered as follows: 1, humpback; 2, fin; 3, blue; 4, sei; 5, Bryde's; 6, gray; 7, common minke; 8, Antarctic minke; 9, pygmy right; 10, right; 11, bowhead; 12, sperm; 13, beaked; 14, bottlenose dolphin; 15, hippopotamus. In several cases, more than one SINE was independently inserted at the same locus, although the insertion sites differed at the nucleotide level. For example, at locus Hump 17, two bands were detected in the bottlenose dolphin, resulting from the insertion of a CD SINE in a common ancestor of cetaceans, followed by an additional insertion of a CDO SINE in a lineage of dolphins (Nikaido et al. 2001a).
SUPPLEMENTAL FIG. 2.—SINE maximum parsimony tree constructed by PAUP* using the data matrix shown in supplemental table 3 (Supplementary Material online). The nodes below the 80% bootstrap values were treated as multifurcations in figure 2.
SUPPLEMENTAL FIG. 3.—Alignment of partial sequences of inconsistent SINE loci. The names of SINE families as well as the subfamilies (in parenthesis) are indicated in boxes. CDs (Cetacean-specific deletions) were characterized as subfamilies of the CHR-2 SINE family (Nikaido et al. 2001a). Direct repeats are highlighted. Identical nucleotides are shown by dots, and deletions are shown by bars. The sequences for this alignment have been deposited in GenBank (accession numbers: AB195472–AB195501).
SUPPLEMENTAL FIG. 4.—Schematic representation of ancestral polymorphisms and incomplete lineage sorting (a) Plus (+) and minus (–) indicate alleles containing or lacking the SINE, respectively. The SINE was amplified in a common ancestor of four species, namely, a, b, c, and d. The alleles containing or lacking the SINE were not fixed completely during the short period (from X to Y). They have since been sorted into lineages and were fixed or lost randomly in each lineage, as the period from speciation to the present time was long enough for this to occur. (b) Although the species tree is the one in which species a and b are sibling species, the gene tree deduced from this locus shows species a and c as a monophyletic group.
SUPPLEMENTAL FIG. 5.—Confirmation of SINE insertion by sequencing and aligning the loci isolated in this study. The middle region of the alignment was abbreviated using Xs. The numberings of the species are followed by that of electrophoresis figure. Direct repeats were highlighted by box. The sequences are not deposited in GenBank because these sequences have been determined just roughly and contain many Ns.
Supplemental Table 1 DNA samples used in this study. The location and the dates of sampling are shown in this table. The samples of unknown location and/or dates are shown by?
Supplemental Table 2 The primers used in this study.
Supplemental Table 3 Data matrix showing the character states for the loci isolated in the present study. 0 = absence, 1 = presence,? = missing. The descriptions of each locus and taxa analyzed in this study are shown in the boxes.
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Acknowledgements |
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For permissions and research support, we thank J. Mead of the U.S. National Museum of Natural History and S. Chivers, A. E. Dizon, and K. Robertson of the Protected Resources Division, National Marine Fisheries Service. This work was supported by a Grant-in-Aid to N.O. from the Ministry of Education, Science, Sports and Culture of Japan.
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Footnotes |
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Dan Graur, Associate Editor
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References |
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