Department of Biological Sciences, Biodiversity and Systematics, University of Alabama, Tuscaloosa
Correspondence: E-mail: serb{at}lifesci.ucsb.edu.
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Abstract |
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Key Words: mitochondrial genome Unionidae Bivalvia gene rearrangement phylogeny
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Introduction |
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Until recently, studies on the gene order of the mt genome have been biased toward chordate taxa, which possess a similar gene arrangement; however in some invertebrate groups, gene order and genome content appears to be less conserved (see review in Boore 1999). The degree of invertebrate mt gene rearrangement appears to be variable across phyla and sometimes within a given phylum. In Arthropoda, the largest animal phylum, gene rearrangement generally appears to be limited to adjacent tRNA genes and areas surrounding noncoding (NC) regions (Boore 1999). For example, between the insect Drosophila and the chelicerate Limulus, gene order varies only in the placement of trnL (taa); there are no rearrangements among protein-coding or ribosomal genes (Staton, Daehler, and Brown 1997; Lavrov, Boore, and Brown 2000). Within specific arthropod lineages such as metastriate ticks (Black and Roehrdanz 1998), anomuran crustaceans (Morrison et al. 2001), and lice (Shao, Campbell, and Barker 2001), there is a greater amount of gene rearrangement that includes not only the translocation of tRNA genes but also protein-coding and occasionally rRNA genes. Although gene rearrangement is relatively limited in arthropods, gene order provides characters with sufficient phylogenetic signal to resolve relationships among distantly related lineages (Boore et al. 1995; Boore, Lavrov, and Brown 1998).
In contrast to the Arthropoda, Mollusca, the second largest animal phylum, displays an extraordinary amount of variation in gene arrangement. Currently, there are 12 complete and three incomplete mitochondrial genomes available on GenBank, representing four of the eight classes within the phylum (table 1). The two best-sampled classes are the gastropods and the bivalves. Four complete gastropod mt genomes are available, representing the pulmonates (Albinaria coerulea [Hatzoglou, Rodakis, and Lecanidou 1995] and Cepaea nemoralis [Terrett, Miles, and Thomas 1996]) and the opisthobranchs (Pupa strigosa [Kurabayashi and Ueshima 2000] and Roboastra europaea [Grande et al. 2002]), and one incomplete data set for the caenogastropod Littorina saxitilis (Wilding, Mill, and Grahame 1999). For bivalves, five complete mt genomes are available, including: Crassostrea gigas (S. H. Kim, E. Y. Je, and D. W. Park, personal communication; GenBank accession number NC_001276); the male and female mitotypes of Inversidens japanensis (M. Okazaki and R. Ueshima, personal communication; GenBank accession numbers AB055624 and AB055625); the male and female mitotypes of Venerupis philippinarum (M. Okazaki and R. Ueshima, personal communication; GenBank accession numbers AB065374 and NC_003354). In addition, there is a nearly complete mt genome for Mytilus edulis (Hoffman, Boore, and Brown 1992) and a partial mt genome for Pecten maximus (D. Sellos, M. Mommerot, and A. Rigaa, personal communication; GenBank accession number X92688).
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Bivalves have the greatest amount of mt gene rearrangement, although this degree of variation may be an artifact of a wider taxonomic sampling in bivalves versus gastropods. Among the currently available taxa, there are very few shared gene boundaries (fig. 1) and gene translocation appears across all gene classes (protein-coding, tRNA, and rRNA). Genome content also varies among bivalve taxa due to apparent gene duplications and losses. Three of the four near-complete mt genome sequences (Crassostrea, Mytilus, and Venerupis) have duplicated tRNA genes. Crassostrea also has a duplicated rrnS, and Venerupis has a second tandem copy of cox2 of a different length.
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Several models have been proposed to explain the mechanisms of gene translocation. The most familiar and accepted is the duplication and loss model, in which a portion of DNA is duplicated during replication, resulting in tandem copies of a gene or genome region. Once a disabling mutation arises in one of the gene copies, that copy is removed from the mt genome or reduced to noncoding sequence during subsequent replications. Recently, it has been proposed that gene loss may be a nonrandom process (Lavrov, Boore, and Brown 2002). There also may be an association between gene translocation and noncoding regions in the mt genome, especially the control region (CR) in vertebrates (Boore 1999). Two other less discussed models are the dimerization of two mt genomes and illegitimate recombination between mt genomes (see description in Boore 2000). The general lack of knowledge for the mechanisms involved in gene translocation and genome rearrangement affects our interpretation of gene order data. Bivalves, with their great diversity, ancient lineages that span the Phanerozoic (Skelton and Benton 1993), and the observed variation in genome content should eventually provide a good system for modeling the mechanisms of gene movement.
In the present study, we determined the complete mtDNA sequence of the bivalve, Lampsilis ornata, a North American representative of the family Unionidae. In addition, we describe the notable features of the Lampsilis mt genome and compare these to other available bivalve taxa. Gene order of Lampsilis is examined in a comparative phylogenetic framework with five other bivalve sequences representing five families and four orders. Our examination of disparate gene arrangement indicates the potential phylogenetic utility of gene order and allows for the reconstruction of ancestral gene order states. Unique mechanisms that may be involved with gene translocation are discussed for Bivalvia.
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Materials and Methods |
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The total length (16.0 kb) of L. ornata mtDNA was amplified by polymerase chain reaction (PCR). In brief, a small (
500 bp) cox1 segment was amplified using primers from Folmer et al. (1994), following the protocol described in Roe and Lydeard (1998). New primers (COI-477F and COI-215R) were designed from sequence of the small cox1 segment, then used in long PCR (LA PCR kit TaKaRa) to amplify the remaining mtDNA region (
16 kb). The resulting large fragment was purified with Microcon concentrators (Millipore) and used as template for cycle sequencing reactions. As a new sequence was generated, it was aligned to the original cox1 sequence fragment to create a continuous DNA strand (contig). New primers were designed from the ends of the contig. To quicken the process of sequencing, additional mitochondrial regions were utilized for primer design to create multiple origins of primer walking. Primers for three of these regions came from the following sources: rrnL (Lydeard, Mulvey, and Davis 1996), rrnS (White, Mcpheron, and Stauffer 1996), nad1 (Serb, Buhay, and Lydeard 2003), and cob (B. Bowen, personal communication). Original primers for this study were also designed from conserved sequence blocks within nad2, nad3, and nad4. Table 2 lists sequence and mt genome position of primers used in PCR and cycle-sequencing reactions. Sequence of the mt genome of Lampsilis ornata has been submitted to GenBank under the accession number AY365193. The voucher specimen is housed in the University of Alabama Unionid Collection under UAUC 3192.
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To examine phylogenetic histories of gene arrangement, gene order was coded and the character states optimized on an independently derived topology. The program MacClade version 4.0 (Maddison and Maddison 2000) was used to parsimoniously assign character states to internal nodes according to information from the topology. The DELTRAN tracing option was chosen to select among the most-parsimonious reconstructions. This tracing method delays changes away from the root, maximizing parallelisms and minimizing reversals. Gene arrangement for each of the molluscan taxa and outgroups was coded by gene boundaries following Boore et al. (1995) and Boore and Staton (2002), where the character was the gene X being either immediately upstream or downstream of the character state gene Y. This method of coding also provides information on transcriptional orientation for each gene relative to its neighbor. For this analysis, two data sets were generated, one including and the other excluding tRNA genes. tRNAs were omitted from one data matrix to determine whether there was any phylogenetic signal associated with mtDNA protein-coding and ribosomal genes, which might be masked by the apparently higher observed rate of translocations of tRNA genes. Genome sequence of taxa with unusual mt genome content, such as an incomplete mt genome sequence or duplication and/or loss of genes, were reevaluated using the methods described above to verify previous authors' findings. Several changes in gene annotation were made for the gene order analysis. In Pecten, the second copy of trnK was reassigned as trnQ, and trnV was newly identified downstream of trnQ. In Crassostrea, five additional tRNA genes were identified, including trnS1, trnS2, a second copy of trnQ, and two undetermined tRNAs that are most likely trnA and trnF. All new putative tRNA genes are illustrated in figure 2. In the F-mitotype of Inversidens, a portion of atp8 was identified between trnD and nad4L based on amino acid similarity (table 3). All tRNA duplications in Mytilus, Venerupis, and Crassostrea and the duplication of rrnS in Crassostrea and cox2 in Venerupis were independently confirmed during our reevaluation. Character states of duplicated, nonadjacent genes (such as trnM in Mytilus) were examined in an attempt to identify the original and duplicated copy of the gene. However, since the majority of duplication codings resulted in autapomorphic character states for the given taxon and did not resolve the question of gene homology, we decided on a conservative approach where nontandem gene duplications were assigned as unknown ("?") in the gene order matrix. Gene arrangement from only the F-mitotype was compared across bivalve taxa under the assumption that gender-specific mitotypes represented orthologous sequences (but see Results and Discussion). A traditional phylogeny of the sampled molluscan classes was used for this study (Salvini-Plawen and Steiner 1996) (fig. 3). Non-molluscan taxa included the arthropod Drosophila melanogaster (Lewis, Farr, and Kaguni 1995) and the annelid Lumbricus terrestris (Boore and Brown 1995). Relationships within Gastropoda were based on Kurabayashi and Ueshima (2000) and Grande et al. (2002). Bivalve relationships in the topology were based on a hypothesis resulting from a total-evidence approach of DNA sequences and morphology (Giribet and Wheeler 2002). Character states were reconstructed using the DELTRAN tracing option in MacClade. Unambiguous reconstructions that support a particular node were noted for bivalve taxa as an estimation of phylogenetic signal in the gene order data. Using only unambiguously reconstructed character states, rather than reconstructing states at equivocal nodes, is a conservative approach of examining gene order data. It is understood that the reconstructed ancestral state for a character is only an estimate and has a level of uncertainty that depends on the phylogeny estimation.
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Results and Discussion |
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tRNA Secondary Structure
Twenty-two sequences that folded into tRNA-like secondary structures and possessed correct anticodon sequences were identified in Lampsilis (fig. 5). Nearly all of the putative Lampsilis tRNAs have a seven-member aminoacyl acceptor arm, a five-member anticodon stem (but see trnA, trnE, and trnG), and a seven-member anticodon loop. The anticodon loop of trnR is unusual, having only six members. A "?" marks the place in the loop that may be missing a base (fig. 5). In Lampsilis, both trnS1 and trnS2 have shortened DHU arm stems, where potential pairing consists of only one (G-T) or two (G-C, T-A) base pairs, respectively. These configurations for trnS are not shown in figure 5. This is not atypical, as the DHU arm of the trnS1 (AGN) is unpaired in many metazoan taxa (Garey and Wolstenholme 1989; Hoffman, Boore, and Brown 1992; Yamazaki et al. 1997; Tomita et al. 2002). The TC arm loop is missing in the Lampsilis trnC, but is present in Mytilus (Hoffman, Boore, and Brown 1992) and Katharina (Boore and Brown 1994); however, the length of T
C arm is variable in gastropods, where either the stem is missing or reduced (Yamazaki et al. 1997). Mispairing between bases in stems is consistent across several taxa. For example, the second base pair in the anticodon stem of trnW has a T-T mispairing in Lampsilis, Mytilus, and Katharina and a T-G pairing in several gastropods (Yamazaki et al. 1997).
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Phylogenetic Implications of Mitochondrial Gene Arrangement in Bivalvia
Two gene order matrices, one including and one excluding tRNA genes, were constructed for the 13 molluscan and two distantly related outgroup taxa. There were 74 characters in the complete matrix and 30 characters when tRNA genes were excluded. Examination of gene arrangement when mapped onto the independently derived molluscan topology (fig. 3) showed several unambiguously reconstructed character states that support the Bivalvia and relationships within the class. The absence of atp8 was reconstructed as the ancestral condition of Bivalvia in both gene order data sets. Another putative ancestral state for Bivalvia was a shared gene boundary between of rrnL-cob when excluding tRNAs from the data matrix. Adjacent trnG and trnN genes are reconstructed as the ancestral character state for the clade containing Crassostrea, Pecten, and Mytilus. This clade was not supported unambiguously when excluding tRNA genes from the analysis. The Unionidae (Lampsilis + Inversidens) was supported by multiple synapomorphies and unambiguous character state reconstructions in both data sets (see below for description), including the presence of atp8 (fig. 6). The placement of Venerupis on the independently derived topology was not supported with either gene order data set. Each of the bivalve taxa has many autapomorphic characters, illustrating the number of unique gene boundaries. Figure 6 lists unambiguously reconstructed ancestral gene order conditions within Bivalvia.
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Maximum-parsimony analysis was conducted on both gene order data sets to generate a topology based solely on gene boundaries. Analysis of the gene order data, including tRNAs, provided a total of 67 parsimony informative characters. The MP analysis resulted in 12 most-parsimonious trees (488 steps). The Mollusca was not recovered as monophyletic, and higher-order relationships among the classes were unresolved (not shown). Both Bivalvia and Gastropoda were recovered as monophyletic groups. Within Bivalvia, relationships differed from the inferred topology (Giribet and Wheeler 2002) by the placement of Venerupis in a polytomy with Crassostrea and Mytilus. Bootstrap values were greater than 50% for three clades: Gastropoda (91%), Pupa+Roboastea (76%), and Lampsilis+Inversidens (96%). Analysis of gene order data, excluding tRNAs, resulted in 25 equally parsimonious trees of 159 steps (not shown). The strict consensus indicates less resolution overall and a monophyletic Bivalvia was not recovered.
Variation Within Unionidae
Gene arrangement between female mt genomes of the North American Lampsilis ornata and Asian Inversidens japanensis are identical except for the region between rrnS and cox1. This region includes three protein-coding (nad2, nad3, and cox2) and eight tRNA (A, E, W, R, M, S1, S2, and H) genes (fig. 7). Interestingly, this region is also variable between the M- mitotypes and F-mitotypes of Inversidens (M. Okazaki and R. Ueshima, personal communication). In all three mt genomes, this variable region contains the largest NC sequence: Lampsilis, 282 bp between trnE and nad2; Inversidens-F, 287 bp between trnW and trnE; and Inversidens-M, 316 bp between trnH and cox1. The association of an NC region with an area of gene translocation has been observed in arthropod taxa (Boore 1999) and may provide a starting point in the identification and characterization of a CR in the Unionidae. Although gene order is more similar between Lampsilis-F and Inversidens-M, phylogenetic analysis of nucleotide sequence variation among the mitotypes recovers the F-mitotypes of Lampsilis and Inversidens as sister taxa supporting the homology of these two genomes under the criteria described by Hoeh et al. (1996) (J. M. Serb, unpublished data). Homology of the F-mitotypes suggest that there has not been a mitotype reversal in the lineage for Lampsilis and Inversidens, and our limited taxonomic sample agrees with the conclusions of Hoeh et al. (1996) that there is a single origin of DUI in the Unionidae lineage.
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What Makes Bivalve Gene Order So Variable?
At this time, the available mt gene order data do not support a particular model of gene rearrangement for bivalves, but it does provide tantalizing clues. Multiple copies of genes exist in extant taxa, but the majority of gene duplications are not tandem. This may be because either the duplication did not form in tandem or the gene translocation occurred subsequently to the duplication event. Our data also show an association of large NC regions within a region of gene rearrangement in unionids and between the rrnS duplication in Crassostrea. Sampling mt genome sequences of more closely related bivalves may provide insight to the mechanism of gene movement by limiting the number of gene translocations while at the same time utilizing gene duplication and loss that frequently occurs in Bivalvia. Bivalves provide a unique opportunity to formulate models by providing historical "snapshots" of gene movement, as duplications are predicted to be intermediate steps in mt genome rearrangements. Also, because of their unique mode of mtDNA inheritance, bivalves may have mechanisms for gene translocation not present in other molluscs (see below).
It is important when examining gene rearrangement to consider the likelihood of the change of character states across the topology. Loss and subsequent regain of a gene would appear to be unlikely, whereas gene duplication and loss of one copy would be more probable with our current understanding of gene translocation models. For example, it is unlikely that atp8 was lost in the ancestor of Bivalvia then regained in the Unionidae lineage. Instead, it is possible that the estimation of phylogeny used in our analysis was incorrect or that taxon sampling for gene order was poor. If Venerupis is actually more closely related to Mytilus (Crassostrea + Pecten) than Unionidae, as indicated in the phylogenetic analysis of the gene order data, then the loss of atp8 would not be a putative ancestral state for the Bivalvia but a synapomorphy for a more limited clade, including Venerupis, Mytilus, Crassostrea, and Pecten. When the topology reflects this relationship, two new unambiguously reconstructed ancestral states (trnW-trnR and trnP-cob) are designated for the Bivalvia. Adding taxa may also affect our estimation of character transformation by filling taxonomic gaps in gene order data for class members. However, if the topology of Giribet and Wheeler (2002) is correct, an alternative hypothesis to explain the distribution of atp8 across Bivalvia is multiple and independent losses of atp8 in various lineages. Again, only after a more thorough taxonomic sampling can this hypothesis be addressed.
The occurrence of doubly uniparental inheritance found in some bivalve lineages may also be involved in gene translocation, gene duplication/loss events, and recombination. The data show that gene content and order can vary between M-mitotypes and F-mitotypes of a species (Inversidens japanensis, M. Okazaki andR. Ueshima, personal communication; GenBank accession numbers AB055624 and AB055625), and it has been previously demonstrated that the mechanism that separates the two mitotype lineages may lapse or has multiple origins in Bivalvia. Phylogenetic evidence from Hoeh et al. (1996) and the existence of heteroplasmic females and homoplasmic males (Quesada, Skibinski, and Skibinski 1996; Garrido-Ramos et al. 1998) suggests that the DUI mechanism may occasionally experience breakdowns where a new M-mitotype lineage may be derived from an F-mitotype (mitotype role reversal or "masculinization"). A reversal can also occur in the opposite direction, where an M-mitotype is "feminized" (Garrido-Ramos et al. 1998). Using the example of atp8, if atp8 is deleted from the F-mitotype early in the bivalve evolutionary history, it could be subsequently "gained" in a later lineage (e.g., Unionidae) if that lineage experiences a role-reversal of the mitotypes. In other words, the M-mitotype, which contains a copy of atp8, would replace the F-mitotype that lacked a copy of that gene. This would result in the presence of atp8 in the newly created F-mitotype and in that bivalve lineage. Hoeh et al. (1996) recognizes at least three independent mitotype reversals across Mytilidae and Unionidae. Although controversial, another possible mechanism associated with changes of gene order and genome content is recombination between sexual mitotypes. Ladoukakis and Zouros (2001) found evidence for homologous recombination between a newly masculinized F-mitotype and a maternally inherited F-mitotype and in the gonadal tissue of a male individual of Mytilus galloprovincialis. A second study by Burzynski et al. (2003) provides additional evidence of recombination in M. trossulus at a different location of the mt genome. Further study will be necessary to establish whether mtDNA recombination is a general phenomenon in bivalves or limited to Mytilus, whether recombination is restricted to particular portions of the mt genome, and whether there is a relationship between recombination and DUI. However, if portions of the mt genome can undergo recombination between mitotypes, then this may be another mechanism for gene rearrangement that may not result in tandem gene duplication.
This study represents the first examination of mitochondrial gene rearrangement in bivalves using a phylogenetic framework. One advantage of this method is the ability to infer ancestral character states for mt gene order in Bivalvia. In addition, the ability to map gene order changes on the phylogeny has the potential to elucidate what mechanisms may be responsible for creating the diversity of gene order variation. Several considerations must be made when attempting to reconstruct gene translocation, including the accuracy of the phylogenetic estimation and the probability of the change of character states across the topology. To better understand and model gene rearrangement in the mitochondrial genomes of bivalves, it will be necessary to include the sequences of additional bivalve mt genomes. Increased taxonomic sampling will allow a more accurate estimation of ancestral character states for mt gene order. It also will be paramount to determine how widespread DUI is within the class and the number of times this mode of inheritance has been compromised. Unrecognized mitotype reversals in the evolution of Bivalvia will confound analysis of gene order data if nonhomologous mt genomes are compared.
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Acknowledgements |
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Footnotes |
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Richard Thomas, Associate Editor
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