Hubbard Center for Genome Studies and Program in Genetics, University of New Hampshire
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In mytilids, the fidelity of this system is sometimes compromised. Occasionally, males can be found which lack the paternal genome (Zouros et al. 1994b
; Saavedra, Reyero, and Zouros 1997
). 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. 1996
). After masculinization, divergence between the paternal and maternal lineages begins de novo (Hoeh et al. 1996
, 1997
). 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. 1997
). Hoeh et al. (1996)
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. 1997
).
In contrast, mitotypes of the two unionid genera sampled cluster together by gender (Hoeh et al. 1996
). 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)
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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. 1997
), and gaps were then inserted into the nucleotide sequences at the corresponding sites. This data matrix was imported into PAUP 4.0b10 (Swofford 2000
) 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. 2001
).
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. 1996
). 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 1988
; Ohara, Dorit, and Gilbert 1989
). 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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. 1996
; Gach and Brown 1997
) 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. 1995
; Pitnick and Karr 1998
; Sutherland et al. 1998
). 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. 1995
; Sutherland et al. 1998
). 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 1997
). Mitochondrially encoded genes are present in the cytoplasm of Drosophila (Amikura et al. 2001
), Xenopus (Kashikawa, Amikura, and Kobayashi 2001
), sea urchin (Ogawa et al. 1999
), planarian (Sato et al. 2001
), and ascidian (Oka et al. 1999
) embryos. In Drosophila, mitochondrial ribosomes are present in the germ plasm and are necessary for proper germ line formation (Iida and Kobayashi 1998
). 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. 1994b
; Saavedra, Reyero, and Zouros 1997
). 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
Abbreviations: DUI, doubly uniparental inheritance.
Keywords: molecular evolution
doubly uniparental inheritance
mtDNA
sex-specific evolution
Unionidae
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
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Amikura R., M. Kashikawa, A. Nakamura, S. Kobayashi, 2001 Presence of mitochondria-type ribosomes outside mitochondria in germ plasm of Drosophila embryos Proc. Natl. Acad. Sci. USA 98:9133-9138
Brown J. R., K. Beckenbach, A. T. Beckenbach, M. J. Smith, 1996 Length variation, heteroplasmy and sequence divergence in the mitochondrial DNA of four species of sturgeon (Acipenser) Genetics 142:525-535
Fisher C., D. O. F. Skibinski, 1990 Sex-biased mitochondrial DNA heteroplasmy in the marine mussel Mytilus Proc. R. Soc. Lond. B Biol. Sci 242:149-156[ISI]
Folmer O., M. Black, W. R. Hoeh, R. Lutz, R. C. Vrijenhoek, 1994 DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates Mol. Mar. Biol. Biotechnol 3:294-299[Medline]
Frohman M., M. K. Dush, G. R. Martin, 1988 Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer Proc. Natl. Acad. Sci. USA 85:8998-9002[Abstract]
Gach M. H., W. M. Brown, 1997 Characteristics and distribution of large tandem duplications in Brook Stickleback (Culaea inconstans) mitochondrial DNA Genetics 145:383-394
Haas F., 1969 Superfamily UNIONACEA Pp. N411N470 in R. C. Moore ed. Treatise on invertebrate paleontology: mollusca 6. Geological Survey of America and University of Kansas, Kan
Hisatomi O., T. Satoh, L. K. Barthel, D. L. Stenkamp, P. A. Raymond, F. Tokunaga, 1996 Molecular cloning and characterization of the putative ultraviolet-sensitive visual pigment of goldfish Vision Res 36:933-939[ISI][Medline]
Hoeh W. R., D. T. Stewart, C. Saavedra, B. W. Sutherland, E. Zouros, 1997 Phylogenetic evidence for role-reversals of gender-associated mitochondrial DNA in Mytilus (Bivalvia: Mytilidae) Mol. Biol. Evol 14:959-967[Abstract]
Hoeh W. R., D. T. Stewart, B. W. Sutherland, E. Zouros, 1996 Multiple origins of gender-associated mitochondrial DNA lineages in bivalves (Mollusca: Bivalvia) Evolution 50:2276-2286[ISI]
Iida T., S. Kobayashi, 1998 Essential role of mitochondrially encoded large rRNA for germ-line formation in Drosophila embryos Proc. Natl. Acad. Sci. USA 95:11274-11278
Kaneda H., J. Hayashi, S. Takahama, C. Taya, K. F. Lindahl, H. Yonekawa, 1995 Elimination of paternal mitochondrial DNA in intraspecific crosses during early mouse embryogenesis Proc. Natl. Acad. Sci. USA 92:4542-4546[Abstract]
Kashikawa M., R. Amikura, S. Kobayashi, 2001 Mitochondrial small ribosomal RNA is a component of germinal granules in Xenopus embryos Mech. Dev 101:71-77[ISI][Medline]
Kumar S., K. Tamura, I. B. Jakobsen, M. Nei, 2001 MEGA2: molecular evolutionary genetics analysis. Version 2.1 Arizona State University, Tempe, Ariz.
Liu H., J. B. Mitton, S. Wu, 1996 Paternal mitochondrial DNA differentiation far exceeds maternal mitochondrial DNA and allozyme differentiation in the freshwater mussel, Anodonta grandis grandis Evolution 50:952-957.[ISI]
Newell N. D., 1969 Subclass PALAEOHETERODONTA Pp. N393N489 in R. C. Moore, ed. Treatise on invertebrate paleontology: mollusca 6. Geological Society of America, University of Kansas, Kan
Ogawa M., R. Amikura, K. Akasaka, T. Kinoshita, S. Kobayashi, T. Shimada, 1999 Asymmetrical distribution of mitochondrial rRNA into small micromeres of sea urchin embryos Zool. Sci 16:445-451[ISI]
Ohara O., R. L. Dorit, W. Gilbert, 1989 One-sided polymerase chain reaction: the amplification of cDNA Proc. Natl. Acad. Sci. USA 86:5673-5677[Abstract]
Oka T., R. Amikura, S. Kobayashi, H. Yamamoto, H. Nishida, 1999 Localization of mitochondrial large ribosomal RNA in myoplasm of the early ascidian embryo Dev. Growth Differ 41:1-8[ISI][Medline]
Pitnick S., T. L. Karr, 1998 Paternal products and by-products in Drosophila development Proc. R. Soc. Lond. B Biol. Sci 265:821-826[ISI][Medline]
Rawson P. D., T. J. Hilbish, 1995 Evolutionary relationships among the male and female mitochondrial DNA lineages in the Mytilus edulis species complex Mol. Biol. Evol 12:893-901[Abstract]
Saavedra C., M.-I. Reyero, E. Zouros, 1997 Male-dependent doubly uniparental inheritance of mitochondrial DNA and female-dependent sex-ratio in the mussel Mytilus galloprovincialis Genetics 145:1073-1082
Sato K., T. Sugita, K. Kobayashi, et al. (15 co-authors) 2001 Localization of mitochondrial ribosomal RNA on the chromatid bodies of marine planarian polyclad embryo Dev. Growth Differ 43:107-114.[ISI][Medline]
Skibinski D. O. F., C. Gallagher, C. M. Beynon, 1994a. Mitochondrial DNA inheritance Nature 368:817-818[ISI][Medline]
. 1994b. Sex-limited mitochondrial DNA transmission in the marine mussel Mytilus edulis Genetics 138:801-809
Sutherland B., D. Stewart, E. R. Kenchington, E. Zouros, 1998 The fate of paternal mitochondrial DNA in developing female mussels, Mytilus edulis: implications for the mechanism of doubly uniparental inheritance of mitochondrial DNA Genetics 148:341-347
Swofford D. L., 2000 PAUP*: phylogenetic analysis using parsimony (* and other methods) Version 4.0b8 Sinauer Associates, Sunderland, Mass.
Thompson J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, D. G. Higgins, 1997 The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools Nucleic Acids Res 25:4876-4882
Zouros E., A. O. Ball, C. Saavedra, K. R. Freeman, 1994a. Mitochondrial DNA inheritance Nature 368:818.[ISI][Medline]
. 1994b. An unusual type of mitochondrial DNA inheritance in the blue mussel Mytilus Proc. Natl. Acad. Sci. USA 91:7463-7467[Abstract]
Zouros E., K. R. Freeman, A. O. Ball, G. H. Pogson, 1992 Direct evidence for extensive paternal mitochondrial DNA inheritance in the marine mussel Mytilus Nature 359:412-414[ISI][Medline]