Evolution of Dinoflagellate Unigenic Minicircles and the Partially Concerted Divergence of Their Putative Replicon Origins

Zhaoduo Zhang1, Thomas Cavalier-Smith2 and Beverley R. Green

Department of Botany, University of British Columbia


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Dinoflagellate chloroplast genes are unique in that each gene is on a separate minicircular chromosome. To understand the origin and evolution of this exceptional genomic organization we completely sequenced chloroplast psbA and 23S rRNA gene minicircles from four dinoflagellates: three closely related Heterocapsa species (H. pygmaea, H. rotundata, and H. niei) and the very distantly related Amphidinium carterae. We also completely sequenced a Protoceratium reticulatum minicircle with a 23S rRNA gene of novel structure. Comparison of these minicircles with those previously sequenced from H. triquetra and A. operculatum shows that in addition to the single gene all have noncoding regions of approximately a kilobase, which are likely to include a replication origin, promoter, and perhaps segregation sequences. The noncoding regions always have a high potential for folding into hairpins and loops. In all six dinoflagellate strains for which multiple minicircles are fully sequenced, parts of the noncoding regions, designated cores, are almost identical between the psbA and 23S rRNA minicircles, but the remainder is very different. There are two, three, or four cores per circle, sometimes highly related in sequence, but no sequence identity is detectable between cores of different species, even within one genus. This contrast between very high core conservation within a species, but none among species, indicates that cores are diverging relatively rapidly in a concerted manner. This is the first well-established case of concerted evolution of noncoding regions on numerous separate chromosomes. It differs from concerted evolution among tandemly repeated spacers between rRNA genes, and that of inverted repeats in plant chloroplast genomes, in involving only the noncoding DNA cores. We present two models for the origin of chloroplast gene minicircles in dinoflagellates from a typical ancestral multigenic chloroplast genome. Both involve substantial genomic reduction and gene transfer to the nucleus. One assumes differential gene deletion within a multicopy population of the resulting oligogenic circles. The other postulates active transposition of putative replicon origins and formation of minicircles by homologous recombination between them.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The chloroplast genomes of algae and land plants are circular molecules, usually a single large circle of approximately 120–200 kbp bearing about 100–250 genes (Palmer 1985Citation ; Reith 1995Citation ; Sugiura 1995Citation ; Turmel, Otis, and Lemieux 1999Citation ). In marked contrast to this generally prevailing genomic organization, the chloroplast genes so far sequenced from the peridinean dinoflagellates Heterocapsa triquetra (Zhang, Green, and Cavalier-Smith 1999Citation ) and Amphidinium operculatum (Barbrook and Howe 2000Citation ) are all found on 2–3 kbp minicircles. Each minicircle contains a chloroplast gene (coding region) and a noncoding region in which two or three parts are highly conserved among minicircles within each species. The noncoding region of these minute chromosomes almost certainly includes a replicon origin and the promoter of the gene, though neither has been functionally characterized (Zhang, Green, and Cavalier-Smith 1999Citation ; Zhang, Cavalier-Smith, and Green 2001Citation ). It might also include sequences important for DNA segregation, but such a function might not be necessary if the minicircle copy number is as high as the 100–1,000 estimated for the analogous mitochondrial single-gene minicircles of dicyemid mesozoa (Watanabe et al. 1999Citation ).

Chloroplast and mitochondrial genomes are simplified relics of the much larger cellular genomes of their cyanobacterial and {alpha}-proteobacterial ancestors (Gray 1999Citation ). The origin of minicircles in dinoflagellates and dicyemids is the most radical evolutionary change in their genomic organization thus far established. The chloroplast minicircles of dinoflagellates and the mitochondrial minicircles of dicyemids are the only known cases of the fragmentation of genomes into completely separate unigenic chromosomes in nature. This makes their origin and maintenance of special evolutionary interest. In order to better understand both processes we have fully sequenced nine further chloroplast minicircular chromosomes from five diverse species of photosynthetic dinoflagellates.

Minicircular chloroplast genes are probably widely present among dinoflagellates, as was first shown by DNA hybridization using chloroplast genes psbA and 23S rRNA. This method revealed minicircle-sized bands on electrophoretic gels of native DNA from a number of dinoflagellate species in addition to those from which complete sequences were obtained: Heterocapsa pygmaea, H. rotundata, and Amphidinium carterae (Zhang, Green, and Cavalier-Smith 1999Citation ). After obtaining similar evidence for several different species, we amplified the psbA and 23S rRNA minicircles by PCR from the genomic DNA of H. niei, H. pygmaea, H. rotundata, A. carterae and the 23S rRNA minicircle only from Protoceratium reticulatum and report their complete sequences here.

We show that the noncoding regions of psbA and 23S rRNA minicircles in these dinoflagellate species are very different from those of H. triquetra and A. operculatum. Sequence comparison indicates that the noncoding regions of both psbA and 23S rRNA minicircles consist of two to four core regions (or cores), very conserved in each dinoflagellate, embedded within variable regions. We discuss the evolution and possible functional significance of these organizational differences among minicircular chloroplast chromosomes for replication or segregation. Although extremely conserved within each species, the cores are very divergent among species. This is typical of concerted evolution in which evolutionary divergence is also accompanied by a molecular process homogenizing all members of a multigene family (Elder and Turner 1995Citation ; Liao 2000Citation ). Concerted evolution was first described for tandemly repeated ribosomal RNA genes in Xenopus (Brown, Wensink, and Jordan 1972Citation ) and has been widely studied for multiple gene families in eukaryotes (see Elder and Turner 1995Citation for reviews). Concerted evolution is also known for several dispersed repeated genes and their flanking noncoding sequences (e.g., Liao 2000Citation ; Meinersmann and Hiett 2000Citation ). However, the concerted evolution of the dinoflagellate core regions appears to be the first example of concerted evolution occurring directly between regions of noncoding DNA flanking nonhomologous genes. As such, it is of considerable evolutionary interest and may also be functionally significant.

The origin of chloroplast gene minicircles in dinoflagellates was a remarkable and unprecedented event in the evolution of chloroplast genomes. We shall present evidence from their broad phylogenetic distribution among dinoflagellates that minicircles originated once only, the initial fragmentation of the chloroplast genome having occurred relatively early in peridinean evolution. We discuss how it may have happened and present two alternative models for its molecular mechanism.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
DNA Extraction
Total DNAs were extracted from the dinoflagellates H. pygmaea (CCMP 1490), H. niei (CCMP 447), H. rotundata (NEPCC D680), and P. reticulatum (NEPCC D535) as described for H. triquetra (Zhang, Green, and Cavalier-Smith 1999Citation ). DNA was extracted from A. carterae (CCMP 1314) by the same method but without glassbead vortexing.

PCR Reactions
Specific dinoflagellate chloroplast 23S rRNA and psbA primers were designed based on the H. triquetra 23S rRNA and psbA sequences; degenerate primers were based on all available chloroplast 23S rRNA and psbA gene sequences, as described elsewhere (Zhang, Green, and Cavalier-Smith 2000Citation ). The specific primer pair 23S1-23S4 and the degenerate primers D23S1-D23S2 (fig. 1 and table 1 ) were used to amplify the noncoding region of minicircular 23S rRNA genes from H. pygmaea, H. niei, H. rotundata, A. carterae, and P. reticulatum. Primer pairs bA1-bA5 or DbA1-DbA5 (fig. 1 and table 1 ) were used to amplify the noncoding region of the psbA minicircles. PCR reactions were carried out for 35 cycles: 94°C for 30 s, 55°C for 30 s, followed by 2 min at 72°C in a GeneAmp PCR system 9600 (Perkin-Elmer). The reaction mixture (50 µl) contained 0.2 mM dNTP, 1x PCR buffer, 0.1–1.0 µg template DNA, 50–200 pmol primer, 2.0 or 2.5 mM MgCl2, and 1.5–2.5 units Taq polymerase (Sigma). Products were purified from low-melting gels or using a purification kit (Amersham-Pharmacia Biotech) and used for sequencing.



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Fig. 1.—Chloroplast psbA and 23S rRNA minicircles of H. triquetra, H. pygmaea, H. niei, H. rotundata, and A. carterae. Gray areas represent coding regions; shaded patterns represent cores of noncoding regions. N and C are the N-terminus and C-terminus, respectively, of PsbA protein. 9GL, 9A, and 9GR are the conserved cores of H. triquetra minicircles. D1, D2, D3, D4, and D5 are the nonconserved regions. In H. niei psbA and 23S rRNA minicircles, the third 90-bp core is at a different location with respect to the other cores. The 23S rRNA minicircle of H. rotundata has two 194-bp cores separated by 20 bp. Locations of specific primer pairs 23S1-23S2, bA1-bA5 and degenerate primer pairs D23S1-D23S2, DbA1-DbA5 are shown on H. triquetra circles

 

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Table 1 Primers for Amplifying the Noncoding Region of psbA and 23S rRNA Minicircles

 
DNA Sequencing
Sequencing reactions were done in a Perkin-Elmer GeneAmp 9600 using the ABI cycle sequencing protocol: 94°C for 5 s, 50°C for 5 s, 60°C for 4 min for 25 cycles. Each reaction contained 2–3 µl Bigdye, 20–30 ng DNA (purified PCR product), 3–5 pmol primer, and distilled water to make up to 10 µl. The sequencing samples were precipitated by adding 1/10 volume sodium acetate (pH 5.2) and 2 volumes 95% ethanol, quenched on ice for 10 min, centrifuged for 20 min, air dried, and analyzed by an ABI 377 automatic sequencer. Sequences were assembled and edited using Staden software (http://www.mrc-lmb.cam.ac.uk/pubseq). Sequences were aligned by Clustal W (Thompson, Higgins, and Gibson 1994Citation ) and manually improved using GDE (Smith 1994Citation ).


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
psbA Minicircles
The noncoding region of the psbA minicircle was amplified by inverse PCR from H. niei, H. pygmaea, H. rotundata, and A. carterae, using outward-directed primer pairs as shown for H. triquetra in figure 1 . This yielded one product from H. rotundata, using primers bA1-bA5 and one product from A. carterae, using degenerate primers DbA1-DbA5 (fig. 1 and table 1 ). However, amplification of H. pygmaea genomic DNA using primer pair bA1-bA5 gave two products of 1.7 and 1.9 kbp. Two products, slightly different in size, were also obtained from H. niei DNA. No PCR product was obtained from P. reticulatum using primers bA1-bA5 or DbA1-DbA5, even though DNA blots hybridized with a psbA probe had suggested that psbA minicircles are present in P. reticulatum (Zhang, Green, and Cavalier-Smith 1999Citation ).

Complete psbA minicircles were assembled from these sequences and the overlapping coding sequences previously determined (Zhang, Green, and Cavalier-Smith 2000Citation ). The size of the psbA minicircles ranges from 2,195 bp in H. pygmaea to 2,311 bp in A. carterae (fig. 1 and table 2 ). In H. pygmaea, the noncoding region of the large psbA circle is 269-bp longer than in the small psbA circle because of insertions, mainly in the D1 and D4 segments (figs. 1, 3 , and table 2 ); curiously the larger circle had shorter D2 and D3 variable regions than the smaller one. These results explain the labeling of doublet bands on genomic DNA blots hybridized with psbA probes (Zhang, Green, and Cavalier-Smith 1999Citation ). Because amplification of the coding region of H. niei gave only one product, whereas amplification of the noncoding region gave two products H. niei probably also has two types of psbA minicircles differing only in the size of the noncoding region, consistent with the labeling of doublet bands on genomic DNA blots hybridized with psbA probes (data not shown).


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Table 2 Characteristics of Minicircular psbA and 23S rRNA Genes of Five Dinoflagellates

 
23S rRNA Minicircles
The noncoding region of the 23S rRNA minicircle was amplified by inverse PCR from genomic DNA of H. niei, H. pygmaea, H. rotundata, A. carterae, and P. reticulatum, using the outward primer pair 23S1-23S4 or the degenerate primers D23S1-D23S2 (fig. 1 and table 1 ). PCR reactions yielded one product from H. pygmaea and H. rotundata, using primers 23S1-23S4 and one product from A. carterae, using primers D23S1-D23S2. However, H. niei amplified using 23S1-23S4 gave two products differing in size by about 0.3 kbp. In P. reticulatum, PCR amplification using D23S1-D23S2 also yielded two products, differing by approximately 0.5 kbp. PCR amplification of the coding region using inward-directed primer pairs gave only one product from H. niei and P. reticulatum (Zhang, Green, and Cavalier-Smith 2000Citation ), indicating that both species have two dissimilar-sized 23S rRNA minicircles: identical in the gene but different in the noncoding region. The 23S rRNA gene in all these dinoflagellates, except P. reticulatum (see subsequently), has the same orientation and organization as in H. triquetra, other algae, and land plants. The smaller PCR products from H. niei and P. reticulatum were cloned and sequenced.

Circular contigs were generated when the sequences of the 23S rRNA genes and associated noncoding regions of each dinoflagellate were assembled (fig. 1 and table 2 ). In general, 23S rRNA minicircles are larger than psbA minicircles (fig. 1 ). The size of the 23S rRNA minicircles also varies more among species, from 2,651 bp in A. carterae to 3,772 bp in P. reticulatum, the biggest thus far sequenced from a dinoflagellate.

Unusual Gene Organization in P. reticulatum 23S rRNA Minicircles
The RNA-specifying region of the P. reticulatum 23S rRNA minicircle is highly similar to that of H. triquetra, with >88% identity, but its gene organization differs strikingly (fig. 2 ). Sequence alignment with 23S rRNA genes of various organisms indicated that the P. reticulatum 23S rRNA gene consists of two fragments (the light gray region and the hatched region in fig. 2 ) that have interchanged their positions without changing the orientation of either part of the gene. This is surprising because all other chloroplast 23S rRNA genes from dinoflagellates and other organisms have the same organization and orientation. However, there are examples of rRNA gene fragmentation in mitochondria, e.g., Chlamydomonas (Boer and Gray 1998Citation ). Moreover, nuclear 28S rRNA is frequently posttranscriptionally fragmented into two or more pieces (six in the trypanosomatid Crithidia and even more in Euglena; Smallman, Schnare, and Gray 1996Citation ). Their large ribosomal subunits can be assembled into a functional unit using rRNA fragments, suggesting that the rearranged P. reticulatum minicircular 23S rRNA gene could also be functional.



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Fig. 2.—Comparison of the 23S rRNA minicircles of P. reticulatum and H. triquetra. Corresponding fragments of the 23S rRNA gene have the same pattern. Curved arrows indicate that the 5'->3' direction is conserved, although the positions of the two fragments in the P. reticulatum gene have been exchanged. Numbers indicate nucleotide number starting from designated zero point. Primer pairs as in figure 1

 
Assembling the sequences of several clones revealed that various indels are present in the noncoding regions of the 23S rRNA minicircles in P. reticulatum. This implies that heterogeneous molecules might be present in the PCR product of the noncoding region. Heterogeneous 23S rRNA minicircles resulting from indels in the noncoding region were also observed in different clones in H. triquetra, despite PCR amplification of the noncoding region yielding only one product (Zhang, Green, and Cavalier-Smith 1999Citation ). Probably, each minicircular chloroplast chromosome of P. reticulatum and H. triquetra is a population of minicircles with a homogeneous coding region but heterogeneous noncoding regions.

Extremely Conserved Cores in the Noncoding Regions
In each dinoflagellate, the noncoding region is very conserved and readily alignable between the psbA and 23S rRNA minicircles. Some motifs within these regions are almost identical in both and are called core regions or cores. For convenience, they are named after whatever single nucleotide run occurs near their center, e.g., the 9G and 9A cores making up the 9G-9A-9G tripartite noncoding region of all nine H. triquetra minicircles (fig. 3 ; Zhang, Green, and Cavalier-Smith 1999Citation ). The sequences of the noncoding regions of different species are apparently unrelated and cannot be aligned (fig. 3b ).



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Fig. 3.—Noncoding regions of chloroplast gene minicircles. (a) Organization of the noncoding regions. Each rectangle represents a core conserved within each species; rectangles with the same pattern are identical or extremely related. HT, H. triquetra; HP, H. pygmaea; HN, H. niei; HR, H. rotundata; AC, A. carterae; D1–D4, variable regions between the conserved cores; N and C: PsbA N-terminus and C-terminus, respectively. Inward-pointing pairs of arrows represent the inverted repeats. Asterisks represent psbA only; see figure 1 for alternative arrangements in 23S rRNA genes. (b) Consensus sequences of cores in the noncoding region of minicircles. Capital letters represent identical bases in psbA and 23S rRNA circles; lowercase, bases only in psbA circle; hyphen, base present in only one circle. 9G, 5G, 6T, 7G, and 5A are core names assigned arbitrarily on the basis of runs of identical nucleotides (bolded) within each core, by analogy with the 9G and 9A cores of HT

 
The noncoding region of H. pygmaea has three identical 94-bp cores (5G) with a run of 5G's near the center (figs. 1 and 3a ). In H. rotundata, the psbA minicircle has three different cores of 111 bp (6G), 194 bp (6T), and 95 bp (6T'), the 95-bp 6T' core being identical to the first 95 bp of the 6T core, i.e., a partial duplication (fig. 3a ). However, the 23S rRNA circle has two complete 6T cores, separated by 20 bp, as well as the 95-bp 6T' core (fig. 1 ). Thus, the 23S rRNA circle has a quadripartite and the psbA circle a tripartite noncoding region. In H. niei, both psbA and 23S rRNA circles have a quadripartite noncoding region, with a core of 169 bp, with a run of 6 T's at its center, and three identical 90-bp cores with a central 7 G's (figs. 1 and 3 ). Interestingly, the single 6T core on the psbA circle lies between two of the 7G cores, but on the 23S rRNA circle all three 7G cores are together (fig. 1 ). There is no obvious sequence relationship between the cores of different species.

The psbA and 23S rRNA circles in A. carterae have a bipartite noncoding region, completely different from the tripartite or quadripartite noncoding regions of the four Heterocapsa species. It consists of a large core of 142 bp and a small 48-bp one (figs. 1 and 3 ). Sequence comparison of the psbA minicircle of A. carterae (CCMP 1314) with the psbA minicircle of A. operculatum (Barbrook and Howe 2000Citation ) revealed that they are identical. Sequence alignments showed that the noncoding regions of the psbA and 23S rRNA of A. carterae circles were highly related to the five A. operculatum circles (petD, atpB, psaA, psbA and psbB), which were assumed to have a 49-bp core (Barbrook and Howe 2000Citation ). Surprisingly, the complete psbA circle sequences (AF206672, ACA311632) of another isolate of A. carterae (CS-21, CSIRO Culture Collection, Hobart, Australia) are not identical to the psbA minicircle of the strain A. carterae (CCMP 1314). These results suggest that A. carterae (CCMP 1314, axenic) and A. operculatum (CCAP 1106, axenic) are probably the same species, despite having been collected from different places, whereas A. carterae (CCMP 1314) and A. carterae (CS-21) are not the same species even though they have the same species name. This suggests that at least one of these three Amphidinium strains is misidentified.

In P. reticulatum, DNA hybridization revealed that the psbA gene may be on both minicircular chromosomes and large molecules, whereas the 23S rRNA gene is only on minicircular chromosomes (Zhang, Green, and Cavalier-Smith 1999Citation ). PCR amplification from genomic DNA using inwardly and outwardly directed 16S rRNA primer pairs indicated that the 16S rRNA gene is also on a minicircle (data not shown). Although the coding region of the psbA gene was successfully amplified, its noncoding region could not be. Because the 23S rRNA minicircle is the only chloroplast gene minicircle completely sequenced from P. reticulatum at the moment, it was not possible to determine its conserved cores in the noncoding region. The noncoding region of the 23S rRNA minicircle of P. reticulatum is unalignable with that of known chloroplast gene minicircles of Heterocapsa or Amphidinium species (see previously).

Short Repeated Sequences in the Noncoding Regions
In each minicircle there are variable spacers between the cores and between them and the coding region (D1–D4, fig. 3a ). The corresponding spacers in psbA and 23S rRNA circles vary in size in each dinoflagellate, and in general, are not conserved between the different minicircles of the same species. At least some of this size variation is caused by short, direct repeat sequences, as found in the D2 region of the nine H. triquetra minicircles (Zhang, Green, and Cavalier-Smith 1999Citation ). In H. pygmaea, the psbA circle has five 26-bp direct repeats in the D2 region, each separated by a few bases. In H. rotundata, the D1 regions of both psbA and 23S rRNA circles have two direct repeats of 20 bp, and the 23S rRNA circle also has two different direct repeats of 20 bp. In H. niei, psbA and 23S circles have two to five repeats of several different sequences (11–51 bp), some of which are shared between the two circles. The P. reticulatum 23S rRNA circle has two 66-bp tandem repeats.

Inverted repeats can form hairpins suggested to have a replication function in the chloroplast genomes of Euglena (Schlunegger and Stutz 1984Citation ) and Chlamydomonas (Wu et al. 1986Citation ). Inverted repeats of 20 and 28 bp were found in the D2 region of H. niei (fig. 4 ) and in the 6G core (111 bp) of H. rotundata (fig. 3a ). Interestingly, a 19-bp inverted repeat was also found in the 9A cores (188 bp) of H. triquetra chloroplast gene minicircles (figs. 3a and 4 ). The inverted repeats are exclusively present in the cores that are not duplicated, i.e., no inverted repeats are found in the three identical cores of H. pygmaea. Two inverted repeats were found in the noncoding region of the P. reticulatum 23S rRNA circle (fig. 4 ). No repeats were found in the minicircles of A. carterae.



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Fig. 4.—Sequences of inverted repeats (bolded) in the noncoding region of minicircles. HT, H. triquetra; HR, H. rotundata; HN, H. niei; PR, P. reticulatum

 

    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Interspecific Variability in Core Organization of the Minicircle Noncoding Region
In H. triquetra, all nine single-gene minicircles (Zhang, Green, and Cavalier-Smith 1999Citation ) share the same tripartite organization of their noncoding regions, as do a family of five selfish minicircles containing gene fragments derived from four of them (Zhang, Green, and Cavalier-Smith 2001Citation ). Thus, the three core regions are well conserved among 14 separate minicircular chromosomes in H. triquetra, whereas the intervening regions (D1–D4) are much less well conserved and in parts cannot be mutually aligned. In A. operculatum, only one core region is conserved across all five minicircles studied by Barbrook and Howe (2000)Citation , although we found two core regions on comparing the two minicircles sequenced here for an A. carterae strain (CCMP 1314), which may actually be the same species. Because H. triquetra and A. operculatum (and the very closely related A. carterae) are only distantly related on rRNA trees (Saldarriaga et al. 2001Citation ), it initially seemed possible that the presence of three core regions on one species and only two in the other might reflect an ancient divergence in minicircle organization.

However, our present results reveal that the basic organization of dinoflagellate minicircle noncoding regions is very divergent even among species belonging to the same genus. Thus, although H. pygmaea has a tripartite organization like that of H. triquetra, all three 5G cores are identical to each other, whereas in H. triquetra only the two flanking 9G cores are mutually related. Heterocapsa niei also has three almost identical cores (7G), as well as a single unrelated 6T core, but the order of the cores is not conserved between the 23S rRNA and PsbA circles. The identical repeats in these three species probably arose by tandem duplication, with subsequent divergence of the variable region coupled with conservation of the identical regions (probably by gene conversion; see subsequently). The fact that in the H. niei psbA circle two of these repeats are separated by an unrelated core sequence means that rearrangements in the order of the cores also occurred. An analogous rearrangement must also have taken place in H. triquetra if the two related flanking cores arose by tandem duplication, not duplicative transposition. In H. rotundata there is evidence of one complete duplication of the 6T core in the 23S rRNA minicircles and a partial duplication of 6T to make the 6T' core in both genes.

The virtually adjacent repetition of the central core in the 23S rRNA H. rotundata arose by a relatively recent tandem duplication. The fact that the repeats are separated by a sequence identical to a part of the D3 region, which is highly variable even among circles in the same species, confirms how recent this duplication is. It is highly probable that the three identical core repeats of H. pygmaea also evolved by tandem duplication, but the high divergence of their flanking regions suggests that this must have been long ago. Heterocapsa is a well-defined apparently monophyletic genus (Daugbjerg et al. 2000Citation ; Saldarriaga et al. 2001Citation .) The lack of conservation of the noncoding regions among its species contrasts strikingly with the conservation within species of the core sequences and their number and relative positions. That there has been ample time for considerable divergence between the D1–D4 regions of Heterocapsa is confirmed by a crude application of a molecular clock. This would make the basal radiation of Peridinea about 2.4 times older than that of Heterocapsa (using fig. 1 of Saldarriaga et al. 2001Citation ). As the fossil record suggests that Peridinea may be only about 80 Myr old (Tappan 1980Citation ; Fensome et al. 1993Citation ), H. rotundata and triquetra may have diverged about 30 MYA from the common ancestor of H. pygmaea and H. niei, whereas H. pygmaea and H. niei may have been diverging from each other for about 20 Myr. Although rates of 18S rRNA evolution are dramatically heterogeneous among different dinoflagellate lineages (Saldarriaga et al. 2001)Citation , the branch lengths for Heterocapsa and the numerous clades nearest to it are relatively short and uniform so these estimates are probably reasonable order-of-magnitude approximations; even if they are in error by several-fold, this would not alter our key point that a very substantial divergence between the species is expected for sequences not subject to strong stabilizing selection or a homogenization mechanism.

The Noncoding Region Probably Includes the Replication Origin
Chloroplast replication origins generally map to noncoding regions in the neighborhood of the rRNA genes (Sears, Stoike, and Chiu 1996Citation ; Kunnimalaiyaan, Shi, and Nielsen 1997Citation ), although the core region of the Chlamydomonas origin partly overlaps a ribosomal protein gene (Chang and Wu 2000Citation ). Replication origins frequently contain predicted stem-loop structures, inverted repeats, and multiple direct repeats, not only in plastids (Sears, Stoike, and Chiu 1996Citation ; Kunnimalaiyaan, Shi, and Nielsen 1997Citation ) but also in a number of systems ranging from bacteria to animals (reviewed in Pearson et al. 1996Citation ).

We previously suggested that the 9A region of H. triquetra could contain the replication origin because it is present both in normal minicircles and in aberrant chimeric ones containing multiple fragments of different genes (Zhang, Cavalier-Smith, and Green 2001Citation ). Inverted repeats were found in the 9A core of H. triquetra, the 6G core of H. rotundata, and adjacent to the 6T core of H. niei (fig. 3a ). Interestingly, inverted repeats are present only on cores without duplicates, not on those with duplicates or triplicates. The fact that inverted repeats were not found in the three identical cores of H. pygmaea or in the noncoding region of A. carterae makes it less likely that they are necessary features of replication origins. On the other hand, inverted repeats have been suggested to be hotspots for recombination (Kawata et al. 1997Citation ), consistent with our model for the recombinational origin of aberrant minicircles in H. triquetra (Zhang, Cavalier-Smith, and Green 2001Citation ).

The conserved cores in the noncoding region of the dinoflagellate minicircles are comparable with the conserved sequence blocks in the control or D-loop region at the origin of replication in animal mitochondria (Quinn and Wilson 1993Citation ). The marked interspecific divergence in Heterocapsa core regions is closely analogous to that seen in comparisons between the conserved sequence blocks of animals that diverged many millions of years ago (Quinn and Wilson 1993Citation ), in keeping with our previous arguments for relatively ancient divergence. In contrast, among Amazona parrots which probably diverged relatively recently (scores of thousands of years), the conserved blocks are identical among different species (Eberhard, Wright, and Bermingham 2001Citation ). The analogy with vertebrate mitochondrial control regions even extends to such details as the stronger conservation in the number of conserved blocks among closer relatives; thus, the differences in conserved block number in early diverging lampreys (Lee and Kocher 1995Citation ), compared with their constancy in tetrapods (Quinn and Wilson 1993Citation ) is closely analogous to the change in core numbers between the most divergent dinoflagellates Amphidinium and Heterocapsa, compared with their greater similarity within Heterocapsa. The similar patterns of sequence conservation and divergence between dinoflagellate noncoding regions and the animal mitochondrial control region may therefore stem from an underlying similarity in replicative function.

The precise number or arrangement of conserved cores per noncoding region cannot be important for replication because in H. rotundata the 23S rRNA circle has an extra copy of one core compared with the psbA circle, and in H. niei the two types of cores are in different orders. Changes in the number of replication control regions have also been observed in animal mitochondria; in snakes both copies have been maintained identically over scores of millions of years, despite very great divergence among species (Kumazawa et al. 1996Citation ), just as we find for Heterocapsa.

Like the tripartite noncoding region of H. triquetra circles, the noncoding regions of H. pygmaea, H. niei, H. rotundata, A. carterae, and P. reticulatum minicircles can all be folded into elaborate secondary structures with various hairpins, stem loops and large loops using DNA fold (http://mfold.wustl.edu/~folder/dna). In all the cases, each core can be a part of a hairpin or a loop, despite the sequences of the noncoding regions being completely different among species. The capacity for secondary structure might, therefore, be important in replication by serving as the replication origin or in DNA segregation (or both) by binding circles to a membrane (Zhang, Green, and Cavalier-Smith 1999Citation ; Barbrook and Howe 2000Citation ).

Concerted Evolution of Cores in the Noncoding Region of Chloroplast Gene Minicircles
Concerted evolution refers to the concerted divergence of members of multigene families, each gene of the family being highly similar or identical within each species but very divergent among different species (Graur and Li 2000Citation ). Concerted evolution was first observed in Xenopus for the spacers of tandemly repeated ribosomal RNA genes (Brown, Wensink, and Jordan 1972Citation ; Hillis et al. 1991Citation ). It is very common in tandemly repeated multigene families, e.g., those encoding histones (Coen, Strachan, and Dover 1980Citation ) and ubiquitin (Nenoi et al. 1998Citation ). It is also observed in dispersed ribosomal RNA operons in bacterial genomes, very likely driven by gene conversion (Liao 2000Citation ). Two mutational mechanisms have been proposed for the concerted evolution of repeated genes: (1) unequal sister chromatid exchange or crossing over (Smith 1976Citation ), which would be effective for generating and maintaining tandem repeats, and (2) gene conversion (Dover 1982Citation ), which can maintain identical sequences dispersed over a chromosome or on different chromosomes within a species. Unequal crossing over may be responsible for the presence of more than one copy of some cores in Heterocapsa species, as well as for the extra copies found in 23S gene circles of H. niei and H. rotundata (fig. 1 ), but duplication through replication error is even more likely. It is difficult to see how unequal exchange could contribute to the maintenance of core identity among different gene circles of the same species, especially because the intervening regions are divergent.

The cores in the noncoding region of chloroplast gene minicircles in dinoflagellates clearly undergo concerted evolution because they are identical or extremely conserved among minicircles in a species but are totally different between species. Does this conservation of core sequences and arrangement reflect selective constraints arising from the probable functions of these regions in replication, transcription initiation, and perhaps chromosome segregation, or is it purely the outcome of the essentially neutral dynamics of gene conversion? We shall argue that the underlying causes of this concerted divergence are likely to involve both gene conversion and selection for similarity (but not identity) between cores; neither of these alone can explain the facts.

We suggest that gene conversion is the primary molecular mechanism maintaining near-identity of the related cores within a species. We previously found evidence for gene conversion in the D2 and D3 regions of H. triquetra minicircles, where there are repeats shared by two or three genic circles but not by all (Zhang, Green, and Cavalier-Smith 1999Citation ). Similar instances of shared identical repeats were found in the D2 regions of a family of five highly rearranged minicircles containing fragments of several genes (Zhang, Green, and Cavalier-Smith 2001Citation ). Furthermore, the sequences of the gene fragments are maintained almost identical to those of the corresponding normal genes, even though it is highly unlikely that any of these fragments is functional. The extra 7G and 6T cores in the 23S minicircles of H. niei and H. rotundata are also unlikely to have been maintained identical to their counterparts in the same circle and in the psbA circles by selection alone because the absence of these extra copies in the psbA circles shows that their presence is not essential.

A key question is whether gene conversion is sufficient explanation for the observed intraspecific homogeneity of the conserved cores or whether they are also subject to selection for functional reasons. The fact that the noncoding regions surrounding the cores (i.e., D1–D4) are not well conserved even between genes of the same species suggests that selection must also be acting on the conserved cores. We cannot conclude that selection is not involved just because the primary structure of the cores is not conserved between species. It may be functionally necessary for each viable minicircle to have a part of its structure in common within a species to bind properly to a common transcription, replication, or segregation machinery. One could argue that both such shared machinery and the noncoding region sequences could be free to evolve relatively rapidly from species to species, subject only to their coevolving to maintain the ability to interact functionally. In another context, it has been suggested that coadaptation of rRNA spacers and transcription factors may contribute to concerted evolution of rRNA genes (Dover 1993Citation ). Though selection may favor partial similarity, it need not require identity. Selection for local similarity would ensure that these parts would always remain sufficiently similar for occasional purely neutral gene conversion to cause a higher level of similarity, even identity, than that required by function alone.

Our recent discovery of a family of five highly rearranged chimeric minicircles in H. triquetra (Zhang, Cavalier-Smith, and Green 2001Citation ) strongly supports such functional coevolution subject to selective constraints, rather than one driven purely by mutation pressure. In marked contrast to the unigenic minicircles reported here, where the genes are intact and their gene products probably functional, the chimeric minicircles contain only fragments of genes (up to four different ones); because they almost certainly have no functional gene products we regard them as selfish circles maintained purely by their replicative ability. However, their noncoding regions have just the same tripartite structure as in the nine fully sequenced functional minicircles of H. triquetra, although several circles have an extra 9G core elsewhere. This strongly indicates that some aspects of the organization of the noncoding region are under strong coevolutionary functional selective constraints (Zhang, Cavalier-Smith, and Green 2001Citation ). Their maintenance even in the putatively nonfunctional chimeric circles makes transcription unlikely to be a key factor. Selection for local similarity would ensure that these parts would always remain sufficiently similar for occasional purely neutral gene conversion to cause a higher level of similarity than required by function alone.

Origin of Chloroplast Gene Minicircles
Phylogenetic analysis suggested that dinoflagellate chloroplasts are related to chromistan and red algal plastids and supports their origin by secondary symbiogenesis (Takishita and Uchida 1999Citation ; Zhang, Green, and Cavalier-Smith 1999Citation ). Recent studies using chloroplast genes (Zhang, Green, and Cavalier-Smith 2000Citation ) and nuclear-encoded, chloroplast-targeted genes (Fast et al. 2001Citation ), together make it likely that dinoflagellate chloroplasts are most closely related to sporozoan plastids, and that the common ancestor of all dinoflagellates was photosynthetic. From nuclear gene phylogenies, at least 10 plastid losses or replacements appear to have occurred within dinoflagellates (Saldarriaga et al. 2001Citation ). The theory that chloroplasts of dinoflagellates and other alveolates were acquired by the symbiogenetic incorporation of a red alga into the common ancestor of alveolates and chromists and that all nonphotosynthetic chromalveolates arose secondarily through evolutionary losses (Cavalier-Smith 1999Citation ) has been strikingly corroborated by Fast et al. (2001)Citation . They found that a duplicate of the host cytosolic glyceraldehyde phosphate dehydrogenase (GAPDH) genes retargeted its protein into the plastid, thereby replacing the original red algal plastid GAPDH not only in dinoflagellates and sporozoa but also in cryptomonad and heterokont chromists. The probability of such duplication and retargeting independently in the four groups is virtually zero; it almost certainly occurred only once in a photosynthetic common ancestor.

Because all other chloroplast genomes, including the relict plastid genomes of Sporozoa (Wilson et al. 1996Citation ; Denny et al. 1998Citation ) are circular molecules with multiple genes, the origin of minicircular chloroplast genes in dinoflagellates must have occurred after dinoflagellates and Sporozoa diverged. The fact that chloroplast gene minicircles are present in several distantly related dinoflagellates indicates that they must have originated relatively early in the evolution of peridinean dinoflagellates (Zhang, Green, and Cavalier-Smith 2000Citation ), i.e., they must have been present in the common ancestor of Heterocapsa, Amphidinium, and Protoceratium. Recent trees constructed using gamma distributions to model intramolecular rate variation suggest that the deep branching of the A. carterae clade and its relatives noted earlier (Saunders et al. 1997Citation ) may have been an artifact of their long branches (Saldarriaga et al. 2001Citation ). Therefore, the hypothesis that ancestral Peridinea already had minicircles (Zhang, Cavalier-Smith, and Green 2001Citation ) needs testing more rigorously by studying chloroplast genome organization in lineages that might have diverged earlier; whether any earlier lineages are extant remains unclear because the explosive radiation of Peridinea impedes the robust resolution of basal branching orders. Nonetheless, gamma trees with a better representation of immediate outgroups are consistent with the deepest divergence for photosynthetic dinoflagellates being between Heterocapsa and Amphidinium (T. Cavalier-Smith, unpublished data) and thus with the presence of minicircles in the last common ancestor of all photosynthetic dinoflagellates.

Irrespective of the timing of minicircle origins, its mechanism is of special evolutionary interest. We propose two models for the origin of chloroplast gene minicircles—(1) Model involving sudden transposition of replication origins: The origin of the unigenic chloroplast gene minicircles from a conventional multigenic circular chloroplast genome could have begun by the duplicative transposition of replicon origin sequences throughout the genome. The resulting chloroplast genome with many randomly dispersed identical origins would have been recombinationally unstable, being highly likely to undergo intra- and interchromosomal recombinations, generating a population of various-sized circles, each able to replicate immediately. This would yield some minicircles with a complete gene and the replication origin (noncoding region), whereas others might carry several intact functional genes sandwiched between partial gene sequences disrupted by insertion of the replication origin. This could eventually form a population primarily of minicircles, especially given selection pressure for smaller molecules, as was suggested for the aberrant minicircles, none of which are larger than normal minicircles (Zhang, Cavalier-Smith, and Green 2001Citation ). (2) Differential deletion model: Because deletion of DNA sequences is very common in evolution, and is an on-going process in all organelle genomes (Martin et al. 1998Citation ; Palmer et al. 2000Citation ; Millen et al. 2001Citation ), the chloroplast gene minicircles might have resulted purely by different deletion within a multicopy population of chromosomes, perhaps mediated by direct repeats (Andersson and Kurland 1998Citation ). The deletions would occur independently on different copies of the chloroplast genome but could be spread in the population of genomes by recombination. Whenever all chloroplast genes except one were deleted, only this gene and the replication region would remain as a novel chloroplast gene minicircle. In either model, there could have been intermediate stages with small circles containing more than one functional gene. Minicircles carrying two genes in tandem were recently discovered in Amphidinium: petB-atpA and psbD-psbE in A. carterae strain CS-21 (Hiller 2001Citation ) and petB-atpA in A. operculatum (Barbrook et al. 2001Citation ). They might be relict intermediates from the original genomic fragmentation into minicircles. However, as the tandem genes are not located close to each other in any conventional plastid genome, these two-gene minichromosomes could have been formed secondarily by the fusion of two unigenic circles and the deletion of one core region, as probably occurred during the evolution of the selfish circles in H. triquetra (Zhang, Cavalier-Smith, and Green 2001Citation ). Studies of a much greater phylogenetic diversity of dinoflagellates should distinguish between these alternatives.

The Amphidinium strains are also reported to contain empty minicircles carrying no recognizable genes but similar in size (1.6–2.5 kbp) to normal single-gene circles (2.3–2.5 kbp) and two-gene circles (2.4–2.7 kbp) (Barbrook et al. 2001Citation ; Hiller 2001Citation ). This supports our suggestion of strong selective pressure for small size, based on a range of 2.2–3.0 kbp for single-gene circles and 2.0–2.3 kbp for the aberrant circles with fragmented genes in H. triquetra (Zhang, Cavalier-Smith, and Green 2001Citation ). Minicircles of the other Heterocapsa species range from 2.2 to 3.4 kbp, with a slightly larger (3.8 kbp) 23S rRNA gene circle in P. reticulatum (table 1 ). Following fragmentation of the conventional plastid genome under either model, selection favoring small size would continually select smaller intermediates and help prevent its reversal by recombination. Thus, high copy number and the intracellular replicational advantage of shorter circles could have been the major selective factors favoring fragmentation of a larger circle into several smaller ones.

In the first model, fewer transpositions and in the second fewer deletions would have been needed if the chloroplast genome had previously been reduced in size by transfer of many genes to the nucleus. We are aware of only 13 minicircular genes in total in all studied dinoflagellates, including those found on two-gene circles (Barbrook and Howe 2000Citation ; Barbrook et al. 2001Citation ; Hiller 2001)Citation , although we have found several additional H. triquetra circles with very weak similarity to other databank sequences (Z. Zhang, E. Filek, K. Ishida, B. R. Green, unpublished data). Although we cannot yet be certain that dinoflagellate chloroplasts have a reduced gene complement, we postulate that many more chloroplast genes were transferred to the nucleus in the ancestor of dinoflagellates than in other photosynthetic eukaryotes. This would have occurred after alveolates diverged from chromists but might have partially preceded the divergence of dinoflagellates and Sporozoa. Low genetic complexity and high copy number may, therefore, be the keys to the origin of minicircles. Massive transfer of a majority of the usual chloroplast genes to the nucleus might also have predisposed them to their radical genomic organization; comparison with mitochondria suggests that evolving unusually small genomes predisposes organelles to major, sometimes bizarre, genomic changes (Zhang, Cavalier-Smith, and Green 2001Citation ).

Broader Evolutionary Implications of Minicircular Gene Organization
The genomic fragmentation that has occurred uniquely in the genomes of dinoflagellate chloroplasts and dicyemid mitochondria (Watanabe et al. 1999Citation ) is quite remarkable. In asking why it occurred in these two instances but not in others, one needs to consider the selective forces that would favor fragmentation into minicircles over maintenance of the single large chromosome of the organelles' ancestor. A number of authors have considered the selective forces that originally favored the joining together of separate genes to make the first chromosomes in the ancestors of bacteria (Cavalier-Smith 1987Citation ; Maynard Smith and Szathmáry 1993Citation ; Cavalier-Smith 2001Citation ). Separate genes may be able to replicate faster, but linked genes are less likely to be lost by unequal segregation when organelles (or cells) divide. In the simulations of Maynard Smith and Szathmáry (1993), there was positive selection for linkage unless the gene copy number was high (i.e., over 40).

Whether selection for unigenic molecules because of a replicative advantage will outweigh that for linkage also depends markedly on how template replication and cell division are coupled (Maynard Smith and Szathmáry 1993Citation ), i.e., on the mechanical and regulatory controls in the cell cycle that affect membrane division and DNA replication and segregation. These factors are likely to have been very different during the origin of cells (Cavalier-Smith 2001Citation ) from those that prevailed during the early evolution of dinoflagellate chloroplasts. Another key difference is that selection acts at three different levels on an organelle: among copies of the genome in an organelle, among organelles within a cell, and among cells (reviewed by Birky 2001Citation ; Rand 2001Citation ). At the suborganelle level, single-gene circles may have a replicative advantage, but at the organelle level, linked genes would be less likely to be lost when organelles divide. The fact that fragmentation has occurred in both dinoflagellate chloroplasts and dicyemid mitochondria means that the balance of evolutionary forces does not invariably force the aggregation of genes into multigenic chromosomes.

The minicircular chloroplast genes in dinoflagellates represent the largest change ever detected in the organization of chloroplast genomes. This unique genomic organization raises many questions for future study, such as the replication and segregation of the minicircular chloroplast genes, the mechanism of copy number control for the different circles, and their variability in organization. Their marked variability among lineages will make them of particular value for research into the evolutionary dynamics of chromosome size and organization and into the interaction between mutation pressures and different levels of selection on the balance between chromosome fragmentation and linkage.

Supplementary Data
The sequences reported in this paper have been deposited in the GenBank database with accession numbers AY004258–004266, AY033400.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank K. Ishida and E. Filek for helpful and enthusiastic discussions and completing several sequences and R. G. Hiller and C. J. Howe for communicating data in advance of publication. B.R.G. and T.C.-S. thank NSERC (Canada) for research grants and T.C.-S. thanks the Canadian Institute for Advanced Research and NERC (U.K.) for fellowship support.


    Footnotes
 
Franz Lang, Reviewing Editor

Present address: Department of Plant Biology, Carnegie Institution, Stanford University Back

Present address: Department of Zoology, University of Oxford, Oxford, U.K Back

Keywords: dinoflagellate chloroplast gene minicircles concerted evolution chromosome fragmentation Back

Address for correspondence and reprints: Dr. Beverley R. Green, Department of Botany, University of British Columbia, 3529-6270 University Blvd., Vancouver, British Columbia, Canada V6T 1Z4. brgreen{at}interchange.ubc.ca Back


    References
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

    Andersson S. G. E., C. G. Kurland, 1998 Reductive evolution of resident genomes Trends Microbiol 6:263-268[ISI][Medline]

    Barbrook A. C., A. C. Howe, 2000 Minicircular plastid DNA in the dinoflagellate Amphidinium operculatum Mol. Gen. Genet 263:152-158[ISI][Medline]

    Barbrook A. C., H. Symington, R. E. R. Nisbet, A. Larkum, C. J. Howe, 2001 Organisation and expression of the plastid genome of the dinoflagellate Amphidinium operculatum Mol. Genet. Genomics 266:632–638

    Birky C. W., 2001 The inheritance of genes in mitochondria and chloroplasts: laws, mechanisms, and models Annu. Rev. Genet. 35:125–148

    Boer P. H., M. W. Gray, 1998 Scrambled ribosomal RNA gene pieces in Chlamydomonas reinhardtii DNA Cell 55:399-411

    Brown D. D., P. C. Wensink, E. Jordan, 1972 A comparison of the ribosomal DNAs of Xenopus laevis and Xenopus mulleri: the evolution of tandem genes J. Mol. Biol 63:57-73[ISI][Medline]

    Cavalier-Smith T., 1987 The origin of cells: a symbiosis between genes, catalysts, and membranes Cold Spring Harbor Symp. Quant. Biol 52:805-824[ISI][Medline]

    ———. 1999 Principles of protein and lipid targeting in secondary symbiogenesis: euglenoid, dinoflagellate and sporozoan plastid origins and the eukaryote family tree J. Eukaryot. Microbiol 46:347-366[ISI]

    ———. 2001 Obcells as proto-organisms: membrane heredity, lithophosphorylation, and the origins of the genetic code, the first cells, and photosynthesis J. Mol. Evol 53:555-595[ISI][Medline]

    Chang C.-H., M. Wu, 2000 The effects of transcription and RNA processing on the initiation of chloroplast DNA replication in Chlamydomonas reinhardtii Mol. Gen. Genet 263:320-327[ISI][Medline]

    Coen E., T. Strachan, G. Dover, 1980 Dynamics of concerted evolution of ribosomal DNA and histone gene families in the melanogaster species subgroup of Drosophila J. Mol. Biol 158:17-35

    Daugbjerg N., G. Hansen, J. Larsen, O. Moestrup, 2000 Phylogeny of some of the major genera of dinoflagellates based on ultrastructure and partial LSU rDNA sequence data, including the erection of three new genera of unarmoured dinoflagellates Phycologia 39:302-317[ISI]

    Denny P., P. Preiser, D. Williamson, I. Wilson, 1998 Evidence for a single origin of the 35 kb plastid DNA in Apicomplexans Protist 149:51-59[ISI]

    Dover G., 1982 Molecular drive: a cohesive mode of species evolution Nature 299:111-117[ISI][Medline]

    Dover G. A., 1993 Evolution of genetic redundancy for advanced players Curr. Opin. Genet. Dev 3:902-910[Medline]

    Eberhard J. R., T. F. Wright, E. Bermingham, 2001 Duplication and concerted evolution of the mitochondrial control region in the parrot genus amazona Mol. Biol. Evol 18:1330-1342[Abstract/Free Full Text]

    Elder J. F. Jr.,, B. J. Turner, 1995 Concerted evolution of repetitive DNA sequences in eukaryotes Q. Rev. Biol 70:297-230[ISI][Medline]

    Fast N. M., J. C. Kissinger, D. S. Roos, P. J. Keeling, 2001 Nuclear-encoded, plastid-targeted genes suggest a single common origin for apicomplexan and dinoflagellate plastids Mol. Biol. Evol 18:418-426[Abstract/Free Full Text]

    Fensome R. A., F. J. R. Taylor, G. Norris, W. A. S. Sargeant, D. I. Wharton, G. L. Williams, 1993 A classification of living and fossil dinoflagellates Micropalaeontology, Spec. Publ. 7

    Graur D., W.-H. Li, 2000 Fundamentals of molecular evolution. 2nd edition Sinauer, Sunderland, Mass

    Gray M. W., 1999 Evolution of organellar genomes Curr. Opin. Genet. Dev 9:678-687[ISI][Medline]

    Hiller R. G., 2001 ‘Empty’ minicircles and petB/atpA and psbD/psbE (cytb559 {alpha}) genes in tandem in Amphidinium carterae plastid DNA FEBS Lett 505:449-452[ISI][Medline]

    Hillis D. M., C. Moritz, C. A. Porter, R. J. Baker, 1991 Evidence for biased gene conversion in concerted evolution of ribosomal DNA Science 251:308-310[ISI][Medline]

    Kawata M., T. Harada, Y. Shimamoto, K. Oono, F. Takaiwa, 1997 Short inverted repeats function as hotspots of intermolecular recombination giving rise to oligomers of deleted plastid DNAs (ptDNAs) Curr. Genet 31:179-184[ISI][Medline]

    Kumazawa Y., H. Ota, M. Nishida, T. Ozawa, 1996 Gene rearrangements in snake mitochondrial genomes: highly concerted evolution of control-region-like sequences duplicated and inserted into a tRNA gene cluster Mol. Biol. Evol 13:1242-1254[Abstract]

    Kunnimalaiyaan M., F. Shi, B. L. Nielsen, 1997 Analysis of the tobacco chloroplast DNA replication origin (oriB) downstream of the 23S rRNA gene J. Mol. Biol 268:273-283[ISI][Medline]

    Lee W. J., T. D. Kocher, 1995 Complete genome sequence of a sea lamprey (Petromyzon marinus) mitochondrial genome: early establishment of the vertebrate genome organization Genetics 138:873-887

    Liao D., 2000 Gene conversion drives within genic sequences: concerted evolution of ribosomal RNA genes in Bacteria and Archaea J. Mol. Evol 51:305-317[ISI][Medline]

    Martin W., B. Stoebe, V. Goremykin, S. Hansmann, M. Hasegawa, K. V. Kowallik, 1998 Gene transfer to nucleus and the evolution of chloroplasts Nature 393:162-165[ISI][Medline]

    Maynard Smith J., E. Szathmáry, 1993 The origin of chromosomes I. Selection for linkage J. Theor. Biol 164:437-446.[ISI][Medline]

    Meinersmann R. J., K. L. Hiett, 2000 Concerted evolution of duplicate fla genes in Campylobacter Microbiology 146:2283-2290[Abstract/Free Full Text]

    Millen R. S., R. G. Olmstead, K. L. Adams, et al. (13 co-authors) 2001 Many parallel losses of infA from chloroplast DNA during angiosperm evolution with multiple independent transfers to the nucleus Plant Cell 13:645-658[Abstract/Free Full Text]

    Nenoi M., K. Mita, S. Ichimura, A. Kawano, 1998 Higher frequency of concerted evolutionary events in rodents than in man at the polyubiquitin gene VNTR locus Genetics 148:867-876[Abstract/Free Full Text]

    Palmer J. D., 1985 Comparative organization of chloroplast genomes Annu. Rev. Genet 19:325-354[ISI][Medline]

    Palmer J. D., K. L. Adams, Y. Cho, C. L. Parkinson, Y.-L. Qiu, K. Song, 2000 Dynamic evolution of plant mitochondrial genomes: mobile genes and introns and highly variable mutations rates Proc. Natl. Acad. Sci. USA 97:6960-6966[Abstract/Free Full Text]

    Pearson C. E., H. Zorbas, G. B. Price, M. Zannis-Hadjopoulos, 1996 Inverted repeats, stem-loops, and cruciforms: significance for initiation of DNA replication J. Cell. Biochem 63:1-22[ISI][Medline]

    Quinn T. W., A. C. Wilson, 1993 Sequence evolution in and around the mitochondrial control region in birds J. Mol. Evol 37:417-425[ISI][Medline]

    Rand D. M., 2001 The units of selection on mitochondrial DNA Annu. Rev. Ecol. Syst. 32:415–448

    Reith M., 1995 Molecular biology of rhodophyte and chromophyte plastids Annu. Rev. Plant Physiol. Plant Mol. Biol 46:549-575[ISI]

    Saldarriaga J. F., F. J. R. Taylor, P. J. Keeling, T. Cavalier-Smith, 2001 Dinoflagellate ribosomal RNA phylogeny suggests multiple losses and replacements of chloroplasts J. Mol. Evol 53:204-213[ISI][Medline]

    Saunders G. W., D. R. A. Hill, J. P. Sexton, R. A. Andersen, 1997 Small-subunit ribosomal RNA sequences from selected dinoflagellates: testing classical evolutionary hypotheses with molecular systematic methods Plant Syst. Evol 11: (Suppl.) 237-259

    Schlunegger B., E. Stutz, 1984 The Euglena gracilis chloroplast genome: structural features of a DNA region possibly carrying the single origin of DNA replication Curr. Genet 8:629-634[ISI]

    Sears B. B., L. L. Stoike, W.-L. Chiu, 1996 Proliferation of direct repeats near the Oenothera chloroplast DNA origin of replication Mol. Biol. Evol 13:850-863[Abstract]

    Smallman D. S., M. N. Schnare, M. W. Gray, 1996 RNA: RNA interactions in the large subunit ribosomal RNA of Euglena gracilis Biochim. Biophys. Acta 1305:1-6[ISI][Medline]

    Smith G. P., 1976 Evolution of repeated DNA sequences by unequal crossover Science 191:528-535[ISI][Medline]

    Smith S. W., 1994 The genetic data environment and expandable GUI for multiple sequence analysis Comput. Appl. Biosci 10:671-675[Abstract]

    Sugiura M., 1995 The chloroplast genome Essays Biochem 30:49-57[ISI][Medline]

    Takishita K., A. Uchida, 1999 Molecular cloning and nucleotide sequence analysis of psbA from the dinoflagellates: origin of the dinoflagellate plastid Phycol. Res 47:207-216

    Tappan H. L., 1980 Dinoflagellates. Pp. 224–462 in The palaeobiology of plant protists Freeman, San Francisco

    Thompson J. D., D. G. Higgins, T. J. Gibson, 1994 CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice Nucleic Acids Res 22:4673-4680[Abstract]

    Turmel M., C. Otis, C. Lemieux, 1999 The complete chloroplast DNA sequence of the green alga Nephroselmis olivacea: insights into the architecture of ancestral chloroplast genomes Proc. Natl. Acad. Sci. USA 96:10248-10253[Abstract/Free Full Text]

    Watanabe K. I., Y. Bessho, M. Kawasaki, H. Hori, 1999 Mitochondrial genes are found on minicircle DNA molecules in the mesozoan animal Dicyema J. Mol. Biol 286:645-650[ISI][Medline]

    Wilson R. J., P. W. Denny, P. R. Preiser, et al. (11 co-authors) 1996 Complete gene map of the plastid-like DNA of the malaria parasite Plasmodium falciparum J. Mol. Biol 261:155-172[ISI][Medline]

    Wu M., J. K. Lou, D. Y. Chang, C. H. Chang, Z. Q. Nie, 1986 Structure and function of a chloroplast DNA replication origin of Chlamydomonas reinhardtii Proc. Natl. Acad. Sci. USA 83:6761-6765[Abstract]

    Zhang Z., T. Cavalier-Smith, B. R. Green, 2001 A family of selfish minicircular chromosomes with jumbled chloroplast gene fragments from a dinoflagellate Mol. Biol. Evol 18:1558-1565[Abstract/Free Full Text]

    Zhang Z., B. R. Green, T. Cavalier-Smith, 1999 Single gene circles in dinoflagellate chloroplast genomes Nature 400:155-159[ISI][Medline]

    ———. 2000 Phylogeny of ultra-rapidly evolving dinoflagellate chloroplast genes: possible common origin for sporozoan and dinoflagellate plastids J. Mol. Evol 51:26-40[ISI][Medline]

Accepted for publication November 27, 2001.