MBE Advance Access originally published online on September 25, 2006
Molecular Biology and Evolution 2007 24(1):74-78; doi:10.1093/molbev/msl132
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Research Articles |
Concerted Evolution of Duplicated Control Regions within an Ostracod Mitochondrial Genome
Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, Ikeda, Osaka, Japan
E-mail: y-ohmiya{at}aist.go.jp.
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Abstract |
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 TOP Abstract IntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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The luminescent marine ostracod Vargula hilgendorfii comprises distinct populations around the Japanese islands. Its mitochondrial DNA is unusual, with duplicated control regions (CRs; CR#1 and CR#2). We determined the sequences of ostracod CRs in 7 different populations. The sequences of CR#1 and CR#2 within any population were extremely similar, above 99.7%; moreover, their derived evolutionary tree indicates that the pairs of CRs have evolved in concert within each mitochondrial genome. These results suggest that an exact replication mechanism controls the concerted evolution of CRs.
Key Words: concerted evolution • duplicate control regions • mitochondrial DNA • Ostracoda • molecular diversity
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Metazoan mitochondrial DNA (mtDNA) is a single circular duplex molecule ranging in size from about 14 to 42 kbp. The mitochondrial gene content is nearly identical across taxa, specifying 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 large noncoding region (LNR) (Boore 1999
). It generally lacks introns and has few noncoding nucleotides, except for the LNR (Attardi 1985). For several metazoans, the LNR has been shown to contain elements that control transcription of mtDNA genes and/or replication of the genome; this region is commonly called the control region (CR). CRs may also contain the initiation sites for the replication of mtDNA genomes. For other metazoans, the largest noncoding region in an mtDNA is generally assumed to be the CR (Boore 1999). The mitochondrial genomes of most metazoa studied to date have only 1 CR. However, those of some snakes (Kumazawa et al. 1996), sea cucumbers (Arndt and Smith 1998), metastriate ticks (Black and Roehrdanz 1998; Campbell and Barker 1999), "Amazona parrots" (Eberhard et al. 2001), fish (Lee et al. 2001), and thrips (Shao and Barker 2003) have duplicated CRs, that is, 2 separate CRs with identical or highly similar nucleotide sequences. The lineage of snakes has had duplicate CRs for over 70 Myr, whereas the lineage of metastriate ticks has had duplicate CRs for over 210 Myr (Kumazawa et al. 1996; Campbell and Barker 1999). Intriguingly, sequence analyses indicate that the 2 CRs of the Australasian Ixodes ticks show branching to have evolved in concert (concerted evolution) in each species. In addition to these, species from 7 other lineages of metazoa also have mtDNA with duplicate CRs (Shao et al. 2005). Duplicate CRs have much potential as phylogenetic markers at fine taxonomic levels, such as within genera, within families, or among families. Thus, the presence of duplicate CRs in the mtDNA genomes of metazoa is an interesting and potentially useful genetic phenomenon. However, it is not known how the nucleotide sequences of duplicate CRs remain identical or very similar over time (Shao et al. 2005).
We have reported that the complete mtDNA sequences of the Sea Firefly Vargula hilgendorfii (Umihotaru in Japanese) also have duplicate CRs with identical or highly similar nucleotide sequences (Ogoh and Ohmiya 2004), suggesting the possibility of concerted evolution of CRs in this species. This ostracod is a member of the subclass Myodocopa, in the phylum Arthropoda. V. hilgendorfii has a body length of about 2–3 mm, large eyes of about 0.2 mm in diameter, and long extending antennae. Their habitat is the sandy bottom of coastal waters at depths of 0.5–5 m. They swim to forage at night and burrow during the daytime. They also have an ovoviviparous life cycle, in which eggs hatch within the uterus and juveniles (A-5 stage) are born (Vannier and Abe 1993). Breeding occurs from spring to autumn in the coastal waters off the major Japanese islands. They do not show characteristic planktonic behavior, even in the juvenile stage, indicating that they cannot cross major marine currents and easily move to other localities. However, they are active swimmers with vigorous appetites, using a keen sense of smell. These features strongly suggest that V. hilgendorfii is a good model for the study of biogeography of marine organisms. We focused on populations of this organism living off the Japanese coast. Their distinctly different groups of mtDNA sequences indicate the divergence of their populations around the Japanese Islands (Ogoh and Ohmiya 2005).
The population structure of V. hilgendorfii raises at least 2 functional and evolutionary questions. Was the concerted evolution of CRs maintained or changed under the divergence of this ostracod? How did mtDNA with duplicate CRs evolve? Here we present analyses of the duplicate CR sequences of V. hilgendorfii collected from 7 locations based on the 5 major populations. Furthermore, we checked the individual difference of duplicate CDs in 2 typical regions. These results clarify the concerted evolution of the CR using a comparison of intraspecific variations of this ostracod.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Sampling and DNA Extraction
Specimens were collected from calm beaches on Noto Island (NTM; 37°08'N, 137°02'E), Tateyama (TYM; 34°59'N, 139°51'E), Abura-tsubo (ABT; 35°09'N, 139°37'E), Tanega-shima (TGS; 30°27'N, 130°58'E), Miyako Island (MYJ; 24°43'N, 125°16'E), Taketomi Island (TTJ; 24°19'N, 124°05'E), and Hateruma Island (HTJ; 24°03'N, 123°46'E), using a bait trap (fig. 1). Collected samples were preserved immediately in 99% ethanol. DNA analysis was based on a single individual from each collection point. Total genomic DNA was extracted from muscle using a DNeasy Tissue Kit (Qiagen, Tokyo, Japan), following the manufacturer's protocol.
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Determination of Partial Sequences
Partial sequences were determined for duplicate CRs from total genomic DNA. Two partial fragments of CRs from the mtDNA genomes were amplified with 2 sets of polymerase chain reaction (PCR) primers: VHF-3734 (5'-TCTCATTGGCCTCTCCCTCTTAGAA-3') with VHR-4746 (5'-ATTACCTCTATTTGACTGTAAAGCTC-3') for CR#1 and VHF-10182 (5'-GCTGGAACCAAAAGGTAA-3') with VHR-11391 (5'-GAGAGATCGTATTGACAGAAAGGTTTGTGACCTC-3') for CR#2. These primers were designed based on the complete mtDNA sequence of this ostracod (Ogoh and Ohmiya 2004
). PCR was carried out in a GeneAmp PCR System 2700 (Applied Biosystems Inc., Foster City, CA). The cycling protocol included an initial denaturation step at 94 °C for 2 min, followed by 35 cycles of 30 s denaturation at 94 °C, 30 s annealing at 50 °C, and extension at 72 °C for 2 min. The PCR was performed in 25-µl reaction volumes with 15.4 µl sterilized distilled water, 2.5 µl 10x ExTaq Buffer (Takara, Tokyo, Japan), 2 µl deoxynucleoside triphospate (25 mM), 1 µl each primer (10 µM), 2 µl 10x PCR Enhancer Solution (Gibco BRL, Gaithersberg, MD), 0.1 µl (0.5 units) ExTaq DNA polymerase (Takara), and 1 µl DNA template. PCR products were separated electrophoretically on a 1% Agarose S gel (Nippon Gene, Toyama, Japan) and stained with ethidium bromide for band characterization under ultraviolet transillumination. PCR products were purified by ExoSAP-IT (USB, OH) and were subsequently used for direct cycle sequencing with a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems Inc.). The sequencing primers were the same as the PCR amplification primers. All sequencing reactions were performed according to the manufacturer's protocol. Sequencing data were collected using a model 3100 Avant Genetic Analyzer (Applied Biosystems Inc.).
Phylogenetic Analysis
The sequences of the 2 CRs determined in this study were aligned using ClustalX with default gap penalties (Thompson et al. 1997). Neighbor-Joining (NJ) and maximum-likelihood (ML) analyses were estimated using MEGA version 3 (Kumar et al. 2004) and fastDNAML (Olsen et al. 1994). Distances were estimated based on Kimura's 2-parameter model (Kimura 1980). Numbers beside internal branches in the constructed trees indicate bootstrap probabilities (>80%) based on 2,000 pseudoreplicates.
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Results |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Duplicated CRs
We have previously reported that V. hilgendorfii is found at 47 localities; these can be divided into 5 major populations (Ogoh and Ohmiya 2005
). In the Nansei Islands, especially, it forms distinct local populations; for example, genetic differences were detected between groups from Tanega-shima and Miyako Islands, although these 2 islands are only about 100 km apart. Therefore, we selected 7 collecting sites based on the 5 major populations to clarify concerted evolution in the CR. We also reported that the cytochrome b (CYTB) sequences of 1 sample are highly homologous to other samples in 1 locality. Therefore, we analyzed the CR sequence of only 1 sample from each of 7 localities and confirmed that the CR sequences of each of 5 samples from 2 localities were conserved (supplementary fig. 1, Supplementary Material online). This figure does not contradict figure 1 and a phylogenetic tree constructed using CYTB sequences (Ogoh and Ohmiya 2005). The duplicated CR#1 and CR#2 sequences from the 7 populations sampled ranged from 777 to 780 bp and from 844 to 856 bp, respectively (table 1), although some CRs contained a gap after alignment. The CR#2 sequence contains the pseudogene (79–80 bp) that is part of the cytochrome oxidase subunit III (COIII) gene and collocates with COIII and CR#1 (Ogoh and Ohmiya 2004) (supplementary fig. 2, Supplementary Material online). The CR#1 and CR#2 sequences within any population sampled showed 99.7–100% similarity (table 1). This shows that CR#1 and CR#2 differed by only 1 or 2 nt in length. On the other hand, the similarities of CR#1 and CR#2 sequences sampled from different populations ranged from 93.5% to 100% (table 2). For instance, CR#1 from locations ABT and TYM was identical, whereas in TTJ and TYM it was only 94.0% similar, indicating that differences were supported by biogeographical background. CYTB shows a similar distribution tendency to these CRs (Ogoh and Ohmiya 2005).
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Phylogenetic Trees of Duplicate CR Sequences
Figure 1 shows the NJ tree of the CR#1 and CR#2 sequences of the 7 populations sampled; this was supported by an ML phylogenetic tree. The NJ tree was consistent in 3 groups of locations: NTM, TGS, ABT, and TYM in the main Japanese islands; MYJ alone; and TTJ and HTJ in the Nansei islands. This classification was consistent with biogeographical relations based on the gene for CYTB (Ogoh and Ohmiya 2005
). The phylogenetic trees also strongly suggest that the duplicated CRs have shown concerted evolution in this ostracod. The sequences of CR#1 and CR#2 within each population are more similar than CRs between populations; for example, CR#1 and CR#2 in the population from MYJ are more alike than are CRs from any other populations sampled. The pseudogene for COIII included in CR#2 also showed high similarity indicating concerted evolution (supplementary figs. 2 and 3, Supplementary Material online). These results suggest that duplications in CR#1 and CR#2 must occur during every mtDNA replication. |
Discussion |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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We have earlier reported evidence suggesting concerted evolution of this ostracod mtDNA (Ogoh and Ohmiya 2004
). Here we demonstrated concerted evolution of CR sequences in 7 different populations from coastal Japan and found that similarities between the CR#1 and CR#2 sequences within individual sample sites were always above 99.7%. The high internal homology between CR#1 and CR#2 showed that the duplications in both sequences must occur during every genome replication. How can we explain this phenomenon? Boore (2000) proposed 3 mechanisms of concerted evolution that may account for mtDNA with duplicate CRs: tandem duplication, dimerization, and illegitimate recombination. The tandem duplication mechanism starts with replication errors, such as imprecise termination and/or slipped-strand mispairing. If these errors occur in the section that has the CR, then the replication will generate an mtDNA with 2 tandem-repeated sections, and each section will contain a CR. Dimerization occurs when 2 linearized monomeric mtDNA sequences join head to tail to form a large circular molecule. A dimeric mtDNA sequence would have 2 CRs and 2 copies of each gene. Illegitimate recombination would occur when a section of 1 mtDNA is cleaved out and then introduced into another mtDNA molecule. If this section contains the CR, then the mtDNA that receives the cleaved section of genome will have 2 CR sequences. Apparently, illegitimate recombination may also cause tandem duplications if the introduced section is next to its counterpart in the receipt mtDNA. However, Shao et al. (2005) concluded that this mechanism of concerted evolution in plague thrips could be ruled out because it could not explain the accurate duplications of CRs in each genome during replication. We agree that it cannot explain concerted evolution of duplicate CRs in this ostracod. Thus, the concerted evolution of CR might be controlled by an exact replication mechanism. Our results suggest a new proposed mechanism of concerted evolution, based on "deleted and duplicated" replication, although we cannot deny the possibility of other replication mechanisms. Figure 2A shows this: during each replication, the old CR#2 is deleted and the duplicated CR#1 is inserted at the same location. This proposed model does not contradict accurate CR duplication. If the process does not occur at every replication, each CR will make a new grouping as shown in figure 2B and differences will accumulate at each replication. However, we cannot explain why or how such an accurate replication occurs.
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The duplicated CRs of snakes and metastriate ticks have occurred over 70 Myr and 210 Myr, respectively. However, for concerted evolution, it is difficult to establish how duplicated CRs can arise among differentiating species over long durations because accumulating mutations in mtDNA complicate analysis. To understand these molecular mechanisms in detail, we need to be able to analyze mtDNA replication within a single species in a restricted geographical location over a short period. These ostracods thus present a unique model to study concerted evolution because they live on a limited area of the Japanese coast and have formed a small yet distinct population within only 10,000 years. The distinctly different groups of V. hilgendorfii ostracods could be a good example of how genomic diversity can arise in a relatively short evolutionary time.
In conclusion, this study is the first detailed analysis of concerted evolution within a single species. CR duplications in the mtDNA genomes of this ostracod occur during replication and retain surprisingly high levels of similarity.
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Supplementary Material |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Supplementary figures 1–3 are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).
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Acknowledgements |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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We thank Y. Nakajima, K. Niwa, K. Kobayashi, C. Suzuki-Ogoh, K. E. Fujimori, N. Wakayama, H. Kohtsuka, M. Saika, Y. Henmi, T. Mori, M. Ito, K. Hashimoto, A. Miru, J. Yamazaki, and N. Shikatani for specimen collection.
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Footnotes |
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Billie Swalla, Associate Editor
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References |
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