Ancient Sex-Specific Extension of the Cytochrome c Oxidase II Gene in Bivalves and the Fidelity of Doubly-Uniparental Inheritance

Jason P. Curole and Thomas D. Kocher

Hubbard Center for Genome Studies and Program in Genetics, University of New Hampshire


    Abstract
 TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
Bivalves of the families Mytilidae and Unionidae show a unique mode of mitochondrial DNA inheritance called doubly uniparental inheritance. In addition to receiving the maternally transmitted mtDNA lineage, males receive a separate mtDNA genome from their fathers. This system is sometimes compromised, in that female genomes are occasionally recruited into the male cycle of inheritance. These masculinization events are common in the Mytilidae but have not been reported in the Unionidae. In order to estimate the age of the male and the female lineages in the Unionidae and to look for evidence of masculinization, we sequenced the junction between the cytochrome c oxidase II gene and the cytochrome c oxidase I gene. The unionid male and female lineages diverged ~450 MYA. There is no evidence for masculinization during this period, suggesting that there are taxon-specific differences in the rate of masculinization. Coincidentally, a 200-codon extension of the COII gene is present in the male genome of the Unionidae and may be responsible for the absence of masculinization.


    Introduction
 TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
Bivalves of the marine family Mytilidae (Pteriomorpha) and the freshwater family Unionidae (Palaeoheterodonta) show a unique mode of mitochondrial DNA (mtDNA) inheritance called doubly uniparental inheritance (DUI) (Fisher and Skibinski 1990Citation ; Zouros et al. 1992Citation , 1994aCitation , 1994bCitation ; Skibinski, Gallagher, and Beynon 1994aCitation , 1994bCitation ; Liu, Mitton, and Wu 1996Citation ). Mothers pass their mitochondrial genome, the maternal lineage, on to all of their offspring. Fathers pass a copy of their mitochondrial genome, the paternal lineage, on to their sons. These two lineages evolve independently, leading to two divergent mitochondrial genomes present within the same organism. Although the female genome is widespread throughout the somatic tissue of males, the male genome tends to be more concentrated in the gonad.

In mytilids, the fidelity of this system is sometimes compromised. Occasionally, males can be found which lack the paternal genome (Zouros et al. 1994bCitation ; Saavedra, Reyero, and Zouros 1997Citation ). Under these circumstances a maternal genome is recruited into the paternal lineage, and the male begins passing this new genome on to his sons (dubbed masculinization to indicate the masculinizing of a female genome; see Hoeh et al. 1996Citation ). After masculinization, divergence between the paternal and maternal lineages begins de novo (Hoeh et al. 1996Citation , 1997Citation ). Masculinization events are most obvious in Mytilus, where some male haplotypes are more closely related to female haplotypes than to other conspecific male haplotypes (Hoeh et al. 1997Citation ). Hoeh et al. (1996)Citation found two cases of such masculinization, one in M. edulis and another in M. trossulus. Based on these results, any case where male haplotypes group with female haplotypes to the exclusion of other male haplotypes has been taken to indicate a masculinization event. Masculinized genomes have become fixed at least six times in the evolutionary history of DUI: four within the Mytilidae, one before the origin of Geukensia, and one before the origin of the Unionidae (Hoeh et al. 1997Citation ).

In contrast, mitotypes of the two unionid genera sampled cluster together by gender (Hoeh et al. 1996Citation ). Segregation of the gender-specific mitotypes has apparently remained faithful since the divergence of these genera 100 MYA. This observation led Hoeh et al. (1997)Citation to raise the question of possible taxonomic differences in the fidelity of DUI.

To better estimate the divergence of the male and female genomes of the Unionidae and to sample for possible masculinization events, we sequenced the junction between the cytochrome c oxidase I (COI) gene and the cytochrome c oxidase II (COII) gene for both male and female lineages in several species of Unionacea. The COI gene is downstream of the COII gene, resulting in a product that includes 300 bp of the 3'-end of the COII gene and approximately 400 bp of the 5'-end of the COI gene. Our data indicate that this divergence is ancient; we propose a model to account for the absence of masculinization in the Unionidae.


    Methods
 TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
Sequencing of Sex-Specific mtDNA
In the case of paternal sequences, DNA was extracted from male gonads undergoing active spermatogenesis. The maternal sequence was derived from female gonads or from mantle tissue. A primer anchored in the COII gene was designed based on sequence from Quadrula refulgens (unpublished data). The primer CO2.2 (5'-CAGTGGTATTGGAGGTATGAGTA-3') is designed to anneal to positions 2413–2434 of the Katharina tunicata mitochondrial genome. The Folmer HCO primer was used to prime synthesis on the COI gene (Folmer et al. 1994Citation ). The reactions consisted of 1x PCR buffer, 2.5 mM MgCl2, 0.18 mM each dNTP, and 0.4 µm each primer. Reactions were cycled at 94°C for 15 s, 45°C for 30 s, and 72°C for 60 s for a total of 30 cycles. PCR using DNA isolated from male gonads produced two bands: one the same size as the maternal product and a band of a size unique to males. These unique paternal bands were excised from the gel, cleaned of contaminating agarose, and cloned using the Promega TA cloning kit. Three clones were sequenced for each species (GenBank accession numbers AF517636AF517643). The PCR products were sequenced using the Amersham cycle sequencing kit as per the manufacturer's instructions. The sequencing reactions were visualized using an ABI 377 fluorescent sequencer.

Sequences for Margaritifera hembeli, Q. refulgens, Quadrula quadrula, Lampsilis teres, and Anodonta implicata were obtained using the aforementioned methods. Amplifications of M. hembeli male gonadal template DNA failed to produce a product different from the female sequence obtained from mantle tissue. This could be because of failure to prime M. hembeli male sequence, rearrangements in the M. hembeli male genome disrupting the adjacency of the COII and COI genes, or M. hembeli not exhibiting DUI. Additionally, amplifications from A. implicata mantle DNA failed (possibly because of the poor quality of the tissue) and unlike the other species sequenced, amplifications from the male gonadal tissue failed to produce both a male and a female product. Again, failure to amplify this genome may be caused by the failure of primer pairs or rearrangements within the A. implicata female genome. Sequences for K. tunicata and Crassostrea gigas were obtained from GenBank (accession numbers U09810 and AF177226). The amino acid sequences were aligned using Clustal X (Thompson et al. 1997Citation ), and gaps were then inserted into the nucleotide sequences at the corresponding sites. This data matrix was imported into PAUP 4.0b10 (Swofford 2000Citation ) and used for phylogenetic analysis. Distances were estimated from the first and second codon positions of the 285 nucleotides of the male-female homologous region of COII. The neighbor-joining (NJ) algorithm and maximum likelihood (ML) estimation were used for tree construction. The Tamura-Nei model was used to correct for multiple substitutions for both distance and maximum likelihood estimation. The data set was bootstrapped (1,000 replicates for NJ and 100 replicates for ML) to determine statistical support. Rates of synonymous and nonsynonymous substitutions were estimated using the modified Nei-Gojobori method of MEGA (Kumar et al. 2001Citation ).

Detection of COII mRNA
RNA was extracted from the A. implicata male gonad using Trizol. The reverse transcriptase (RT) reaction was primed using a poly-T primer with an 18-bp unique sequence on the 5'-end of the poly-T (Hisatomi et al. 1996Citation ). A separate control reaction was set up including all ingredients except the RT. The RT reaction was incubated for 1 h at 42°C and then at 70°C for 5 min to inactivate the enzyme (Frohman, Dush, and Martin 1988Citation ; Ohara, Dorit, and Gilbert 1989Citation ). PCR was performed using a primer that annealed to the unique sequence of the poly-T primer and a primer that was at the 3'-end of the homologous region of COII (CO2.8, 5'-AATCATTCGTTTATGCC-3'). Only the RT treatment produced a visible product when run out on an agarose gel. This band was excised, cleaned of agarose, and cloned using the Promega TA cloning kit. Three clones were sequenced; all showed polyadenylation of the complete COII product.


    Results
 TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
Phylogenetic analysis of the male-female homologous region of COII indicates an ancient divergence of these genomes (fig. 1 ). The reciprocal monophyly of the male and female lineages is strongly supported by bootstrapping for both NJ and ML analyses. This divergence was previously dated to 100 MYA (Hoeh et al. 1996Citation ). To better estimate the age of the divergence we analyzed the female sequence from M. hembeli (Palaeoheterodonta: Margaritiferidae) and C. gigas (Pteriomorphia: Ostreidae). The inclusion of M. hembeli with the female lineage indicates that the male-female divergence preceded the divergence of the Margaritiferidae and Unionidae and places a minimum bound of 213 MYA on this divergence (Haas 1969Citation ) (fig. 1 ). Crassostrea gigas branches off before the divergence of the male-female lineages and, therefore, places a maximum bound of approximately 500 MYA on the male-female divergence (the Palaeoheterodonta diverged from other bivalve lineages in the middle Cambrian) (Newell 1969Citation ). By calibrating divergence estimates with the fossil record, we estimate that this male-female divergence occurred more than 450 MYA.



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Fig. 1.—NJ (a) and ML (b) phylogenetic reconstruction of male and female lineages. Genetic distance for each branch is shown above the branches and bootstrap support is shown below the branches. Distances smaller than 1% are not shown. Both NJ and ML bootstrap support for the ancient separation of the male and female lineages is very strong and rejects the hypothesis of masculinization within these unionids. Katharina tunicata is used as an outgroup

 
PCR amplification of the COII-COI junction also revealed an approximately 600-bp insertion within the paternal genome that is absent from the maternal genome. We have found this sex-specific polymorphism in multiple individuals of the same species and in ten additional species (Lampsilis claibornensis, Lampsilis radiata, Amblema plicata, Obliquaria reflexa, Plectomerus dombeyanus, Glebula rotundata, Pyganodon grandis, Villosa lineosa, Utterbackia imbecillis, and Fusconaia flava) where the male genome could be amplified by the PCR. There is the possibility that the sequences identified could be highly divergent nuclear pseudogenes of the female sequence or nuclear pseudogenes of male sequence; however, male-specific length products were only amplified from male gonadal tissue and were not present in female gonadal or mantle tissue. The extra male sequence is a 200-codon extension of the COII gene (fig. 2 ). In the four taxa sequenced, the reading frame of the COII gene extends through this region and terminates with a stop codon just before the beginning of the COI gene. Comparison of the extension between the two most closely related male sequences reveals patterns of evolution consistent with the homologous protein-coding region of COII. In both regions, the rate of substitution at third codon positions is almost 10 times that at first codon positions and over 20 times the rate at second codon positions (table 1 ). The rate of synonymous substitutions is approximately 10 times greater than nonsynonymous substitutions (table 1 ).



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Fig. 2.—The alignment of the putative amino acid sequence of the 3'-end of COII. M indicates male lineage and F indicates female lineage. Sequences end with the termination codon (symbolized by an open circle). The extension sequences are highly divergent with a few areas of conserved amino acid identity. "*" indicates fully conserved; ":" indicates strong group conserved, and "." indicates weak group conserved sites, respectively. After the female termination codon these symbols reflect male sequence identity only. Strong and weak groups are defined in Thompson et al. (1997)Citation

 

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Table 1 Genetic Distances for Male Quadrula Sequences for the Homologous Region and the Extension

 
The alignments of the male-specific extension indicate that the extension is evolving more rapidly than the male-female homologous region (fig. 2 ). Distance estimates between male genomes for the male-female homologous region range from 1.6% to 15%; however, in the extension, these estimates range from 4% to 108% (table 2 ). Blast analysis of the extension reveals no significant similarity with any sequences in GenBank.


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Table 2 Genetic Distances for Male Sequences for the Homologous Region and the Male Extension

 
To verify that this extension is expressed as a part of COII, we used RT-PCR to amplify polyadenylated COII mRNA from A. implicata. The use of the poly-T primer was necessary to verify that this extension was processed as part of the COII gene because the mitochondrial genome is transcribed as a single strand. Using a primer anchored in the homologous region of COII and a unique primer sequence anchored to the 5'-end of the poly-T, RT-PCR produced a product of the expected size. Cloning and sequencing of the product revealed a polyadenylated sequence that was identical to the extended sequence and terminated with the termination codon (TAG) shown in figure 2 .


    Discussion
 TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
Previous studies of DUI in the Unionidae have failed to find masculinization events, which contrasts with the marine mussels where masculinization events occur with measurable frequency. In their survey of marine and freshwater bivalves, Hoeh et al. (1996)Citation found five masculinization events within the marine bivalves, yet none in the freshwater bivalves. Our results bring the number of unionid genera sampled to five and the total number of species sampled to seven. This is over three times as many species as sampled from the single genus Mytilus, in which there have been numerous masculinization events. In concordance with this, the unionid mitotypes are two orders of magnitude older than the mytilid mitotypes. Hoeh et al. (1996)Citation set a minimum divergence for these mitotypes at 100 MYA. The two families represented in our study, the Unionidae and Margaritiferidae, diverged 213 MYA in the Triassic, establishing this as a minimum bound on the male-female divergence (Haas 1969Citation ). Calibrating this divergence with the fossil record and then applying that calibration to the male-female split, we arrive at an estimate of 450 MYA for the actual divergence of these two lineages. This places the divergence of these lineages close to the origin of the Palaeoheterodonta in the middle Cambrian; thus, it is likely that these lineages have remained separate throughout the evolution of the Unionacea. In stark contrast, the most common haplotypes in the M. edulis species complex, as well as Geukensia, are estimated to have diverged only 5.3 MYA (Rawson and Hilbish 1995Citation ; Hoeh et al. 1996Citation ). The faithful maintenance of these separate lineages in the freshwater bivalves over 450 Myr and the frequent breakdown of DUI in the marine bivalves leads us to conclude that the fidelity of DUI differs among the taxa. We hypothesize that there are mechanisms preventing masculinization in the Unionidae, rather than mechanisms promoting masculinization in the mytilids.

The absence of masculinization in the Unionidae correlates with the presence of a male-specific 200-codon extension of the COII gene. It is possible that this extension has no function and is simply extraneous DNA, but we reject this hypothesis on the following grounds. In all the four taxa sequenced the extension is in frame with the COII gene, and stop codons are only present near the initiation codon of the COI gene. As is common in the protein-coding genes of the mitochondria, the positional rates of substitution fall in the 3rd >> 1st > 2nd pattern, and synonymous substitutions are an order of magnitude greater than nonsynonymous substitutions. Lastly, we detected in the male gonad a polyadenylated mRNA transcript of the COII gene that includes the extension. Based on these observations, we conclude that this extension is protein-coding and functional.

The mitochondrial genome of metazoans is often very compact and devoid of any extraneous DNA. Although the genome can vary significantly in size, this is generally the result of duplications involving the D-loop (e.g., Brown et al. 1996Citation ; Gach and Brown 1997Citation ) rather than significant extensions of unique sequence. The alignments with Katharina, Crassostrea, and Loligo indicate that the length of the unionid female COII gene is similar to these COII genes, which excludes the possibility that there has been a contraction of the unionid female COII gene. Also, there has been ample opportunity for loss of the protein-coding nature of the extension given the high rate of evolution in this region of the gene (tables 1 and 2 ). The conservation of this extension for over 100 Myr argues in favor of functionality of this extension.

Elucidation of the function(s) of the extension may help us to understand the evolution of DUI. Hypotheses about function can be divided into three broad categories: a role within the male mitochondrion itself (within mitochondria), a direct role in the survival of the male mitochondrion (within cells), and a function outside of the male mitochondrion (within organisms). These hypotheses and how each might lead to an absence of masculinization are outlined below.

The function of this extension could be within the sperm mitochondrion itself. This must be considered the current null hypothesis because of the normal localization of COII in the inner mitochondrial membrane. The possible functions of this extension in the mitochondrion are numerous, including the obvious function of mitochondrial respiration. However, the rate of evolution of this extension is substantially greater than the rate of evolution of the homologous region of the COII gene. If the function of the extension is limited to within the mitochondrion, the extension could be the direct cause of the absence of masculinization only if it provides a selective advantage over the female genome at some part of the life cycle. It is important to note that in this case the extension functions within the male mitochondrion, but selection at various levels for the presence of the extension could be maintaining the fidelity of DUI.

This extension might be directly involved in intracellular interactions determining the survival of the male mitochondrion. In other organisms, including bivalves, it has been shown that upon fertilization the sperm-derived mitochondria are targeted for elimination from the embryo (Kaneda et al. 1995Citation ; Pitnick and Karr 1998Citation ; Sutherland et al. 1998Citation ). In Mytilus this destruction is sex-specific; the sperm-derived mitochondria survive in embryos destined to be males, whereas in females these mitochondria are destroyed. Elimination of sperm-derived mitochondria appears to involve nuclear encoded genes (Kaneda et al. 1995Citation ; Sutherland et al. 1998Citation ). The COII extension could be involved in blocking elimination to ensure survival of the male mitochondrion. Alternatively, the extension could play a role in the segregation of male mitochondria to the gonad. In either case, it should be possible to detect the protein product of the extension outside of the inner mitochondrial membrane. The protein product should also directly interact with the factors responsible for any of these processes. Again, a selective advantage is necessary for the extension to be the direct cause of the absence of masculinization.

Finally, the extension might function outside of the inner mitochondrial membrane in a way not directly related to survival of the mitochondrion. In this case, expression again might be observable outside of the inner mitochondrial membrane. However, the protein product could be involved in a number of other pathways, including sperm structure, species-specific recognition, or gonadal development (Saavedra, Reyero, and Zouros 1997Citation ). Mitochondrially encoded genes are present in the cytoplasm of Drosophila (Amikura et al. 2001Citation ), Xenopus (Kashikawa, Amikura, and Kobayashi 2001Citation ), sea urchin (Ogawa et al. 1999Citation ), planarian (Sato et al. 2001Citation ), and ascidian (Oka et al. 1999Citation ) embryos. In Drosophila, mitochondrial ribosomes are present in the germ plasm and are necessary for proper germ line formation (Iida and Kobayashi 1998Citation ). Interestingly, if the extension plays a role in male gonadal development, fitness differences between genomes are not required to maintain the fidelity of DUI.

These hypotheses all require some type of functional inequality between the male and female genomes. Testing these hypotheses will require elucidation of the function of this extension and quantification of male fitness with and without the male mitochondrial genome. We have not observed any male haplotype negative individuals in our surveys of natural populations; however, breakdown of DUI has been observed in pair crosses of Mytilus (Zouros et al. 1994bCitation ; Saavedra, Reyero, and Zouros 1997Citation ). Similar pair crosses within the Unionidae may be successful in generating the intermediate male phenotypes required for masculinization. Until these phenotypes are available, identification of the temporal and spatial expression of the extension will be essential.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
We would like to thank Todd Streelman, Karen Carleton, Patrick Danley, Masahiro Okazaki, and Eleftherios Zouros for comments on a precursor manuscript and A. Eyre-Walker and two anonymous referees for comments on this manuscript. J.P.C. would like to thank Sarah Curole for support. This study was funded by a grant from the Conchologists of America.


    Footnotes
 
Adam Eyre-Walker, Reviewing Editor

Abbreviations: DUI, doubly uniparental inheritance. Back

Keywords: molecular evolution doubly uniparental inheritance mtDNA sex-specific evolution Unionidae Back

Address for correspondence and reprints: Jason P. Curole, Hubbard Center for Genome Studies, Environmental Technology Building, 35 Colovos Road, University of New Hampshire, Durham, New Hampshire 03824. jcurole{at}cisunix.unh.edu Back


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 Discussion
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Accepted for publication April 12, 2002.