Two Events Are Responsible for an Insertion in a Paternally Inherited Mitochondrial Genome of the Mussel Mytilus galloprovincialis

Frequent nonhomologous recombination has been previously postulated to explain the 1045-bp insertion in one mitochondrial sperm-transmitted haplotype of Mytilus galloprovincialis. Such recombination would lead to the disruption of gene order and so the existence of a specific mechanism for maintaining the same gene order in both mitochondrial genomes of Mytilus has been proposed. Here the simpler explanation of the observed structure, involving a tandem duplication and a deletion, is presented. Their occasional occurrence in Mytilus mtDNA proves the similarity, not the difference, between animals with and without DUI.

SPECIES of the mussel family Mytilidae have two mtDNA genomes, one transmitted through the egg (the F genome) and one transmitted through the sperm (the M genome) (SKIBINSKI et al. 1994a,b; ZOUROS et al. 1994a,b). The system is known as doubly uniparental inheritance (DUI). In a recent study published by MIZI et al. (2006), authors give what they claim to be the most parsimonious explanation of the origin of an unusual sperm-transmitted haplotype of the Mytilus galloprovincialis mussel originally described by MIZI et al. (2005). The haplotype in question has one large insertion of identifiable composition. Authors originally interpreted it as the fragment of trnN, part of the control region (CR), complete trnY, part of cob, and the fragment of trnG (GenBank AY363687 bases 14,968–16,012). However, due to high homology between the fragments at 14,912–14,978 and at 15,957–16,023 (both comprising mostly fragments of trnG and trnN), it is equally correct to say that the insert consists of a 3' fragment of trnG, 5' fragment of trnN, a large part of the CR, complete trnY, and the 5' fragment of cob (bases 14,912–15,965). The proposed mechanism that produced the insertion involved three steps: short tandem duplication comprising exactly two tRNA genes, a nonhomologous recombination between two different mtDNA genomes, and a deletion. Here I propose another, more parsimonious explanation of the mechanism that might have generated this insertion and show that no more than two events are necessary for it. The first event involved a duplication (Figure 1A) in a typical genome (a) that produced a long tandem repeat spanning the region from trnG to cob (genome aa). The second event was a deletion (Figure 1B) that removed from the first copy of the tandem repeat the following sequences: the large part of trnN, trnE, trnC, trnI, UR6, trnQ, trnD, rnl, and the beginning of the CR producing the genome (d) with the 1045-bp long insertion. The alignment of the two parts of extant genome d spanning the region of duplication and deletion allows identification of the exact insertion and deletion points (Figure 1C). The insertion point was situated within trnG gene. It was located within the nine-nucleotide motif TAAATAACA repeated at the beginning and at the end of the insertion. Motifs repeated at boundaries of tandem repeats are not unusual—they may be involved in, or be a product of, their generation. Here the motif was almost certainly present in the genome a as it is a part of both complete trnG and cob and thus may have been involved in the generation of the repeat. This motif is the same as one of the motifs postulated by MIZI et al. (2006) as one of nonhomologous recombination points. The other nonhomologous recombination point was postulated to occur at, or close to, the seven-nucleotide AGATAGT motif. According to my interpretation, this second motif marks the beginning of the deletion and is also present in the 3' part of the deleted fragment. It is unlikely that the homology between sequences at the deletion boundaries would be necessary for the deletion to occur, and this seven-nucleotide homology could be a pure coincidence.

View larger version (33K): In this window In a new window Download PPT slide   FIGURE 1.— The two events that produced the insertion. The single-letter amino acid code denotes tRNA genes; CR is the major control region; unassigned region 6 (UR6) is a 16-bp noncoding sequence; and rns, rnl, and cob indicate small rRNA, large rRNA, and cytochrome b genes. (A) Tandem duplication: a is the standard gene order; aa is the gene order resulting from tandem duplication of a region boxed in a. Two copies of each named sequence part are numbered 1 and 2, regardless of the completeness of each copy. (B) Large deletion: open sections indicate the first copy and shaded sections indicate the second copy of the repeat along with the rest of the genome. Genome d has the gene order of the examined genome. (C) The schematics of an alignment of the first and second repeats. Bars indicate the three parts of alignment shown in detail. Boldface type indicates a sequence match. Boxed nucleotides represent the motifs discussed in text. The numbers refer to nucleotide positions in the complete sequence of the M genome of M. galloprovincialis containing the insertion.

 
Several arguments can be offered in support of the proposed scenario for the insertion. This scenario calls neither for the involvement of more than one genome in the process nor for nonhomologous recombination between them, yet it solves the order of genes in the resulting genome as well as the original hypothesis of MIZI et al. (2006), which does both. The relatively low genetic distance between the insertion and the rest of the genome (0.005) is better explained under this scenario. Tandem duplications are usually very similar sequence-wise, whereas similarity between arbitrary genomes involved in recombination is an independent condition that would have to be met. Relatively high distances (0.087 on average, according to MIZI et al. 2006) between M genomes found at the present time in M. galloprovincialis make recombinational origin of this insertion even less probable. The only weakness of this explanation is the postulated extinct genome aa having very large duplication (2770 bp), much larger than the duplication postulated by MIZI et al. (2006) in the extinct genome b. However, tandem duplications are quite frequent in animal mtDNA and duplications much >2770 bp have been shown to exist in other species, sometimes also participating in intraspecies polymorphism—for example, in fish (GACH and BROWN 1997), lizards (MORITZ and BROWN 1987), and scallops (GJETVAJ et al. 1992). In Mytilus there have also been reports on larger-than-typical mitochondrial genomes (WENNE and SKIBINSKI 1995) also having multiplicated CR, trnY, and part of cob (BURZYSKI et al. 2006).

Duplications may be involved in the evolution of mtDNA (LAVROV et al. 2002), but to be effective at changing the gene order they have to be both very large and relatively frequent. A small evolutionary distance between sequences of repeats permitted MIZI et al. (2006) to postulate the generation of such rearranged genomes at a high rate. However, tandem repeats in mtDNA may have a tendency to maintain the sequence identity throughout evolution (KUMAZAWA et al. 1996; EBERHARD et al. 2001; ABBOTT et al. 2005). Therefore, the distance between sequences of repeats is not necessarily a good measure of the time since their emergence. A possibly better measure, giving at least a reliable upper estimate, would be the distance between extant genomes a and d. Unfortunately, identifying the example of extant genome a is not trivial. Introgression of mtDNA is so common in Mytilus mussels that proper taxonomic identification of an individual does not guarantee the identity of its mtDNA. Population studies have shown that M-type mitochondrial genomes have not crossed the Atlantic since 1 MYA (RIGINOS et al. 2004). It is generally accepted that there have been two earlier separation events (RAWSON and HILBISH 1995). The first one occurred after the opening of Barents Strait (3.5 MYA) and resulted in the speciation of M. trossulus and M. edulis from their ancestor. The second one was associated with the closing of the Mediterranean Sea during the Pleistocene (>1 MYA), resulting in the emergence of M. galloprovincialis from M. edulis. Each of those events must have left signatures in both M- and F-genome mtDNA. The signature should be more evident within the M lineage since the M genome apparently evolves faster (STEWART et al. 1995) and is not as prone to introgression (RIGINOS et al. 2004). There now should be at least four distinct clades of M-genome mtDNA in the M. edulis species complex: American M. trossulus, American M. edulis, European M. edulis, and M. galloprovincialis. Building a phylogenetic tree from published sequences confirms the existence of those four clades. For the region in question, sufficiently complete GenBank records representing the three most recent clades are the following: for American M. edulis: AY350792, AY350791 (CAO et al. 2004), and AY823623 (BRETON et al. 2006); for European M. edulis: AY115482 (BURZYSKI et al. 2003) and AY350794 (CAO et al. 2004); and for M. galloprovincialis: AY629163 and AY629164 (MIETANKA et al. 2004). The average Kimura two-parameter distance between the American and European M. edulis is 0.031 (SE = 0.006) in the region corresponding to the insertion. The average distance between the rearranged sequence discussed here and the closest true M. galloprovincialis sequences is 0.028 (SE = 0.006). Assuming the same rate of evolution for all M genomes and using trans-Atlantic separation as a dating point, I conclude that the duplication must have occurred not earlier than 750 KYA (on the basis of the distance from the M. galloprovincialis clade), and no later than 100 KYA (on the basis of the between-repeat distance). Even though this estimate is very rough, it indicates that the divergence between the rearranged genome and its ancestor may be much greater than the average distance between M. galloprovincialis M genomes, as postulated by MIZI et al. (2006). Consequently, such large duplications potentially disrupting gene order may be relatively rare.

The original interpretation implies that arbitrary nonhomologous recombination, a process that would easily lead to dramatic changes in gene order, frequently occurs in mtDNA of M. galloprovincialis, necessitating particularly strong selection for gene order to explain the stability of M-genome structure in DUI animals. But, in my interpretation, no such postulate is needed. According to RAWSON and HILBISH (1995), the separation of M and F lineages in the M. edulis species complex occurred 5.3–5.7 MYA. Partial duplications involving large parts of coding sequences could have happened only a few times during that period. Since such duplications are less likely than arbitrary nonhomologous recombination to lead to a dramatic change of the gene order, there is no reason to expect a different order of genes in M and F genomes. Hence the selection for the same gene order in animals with DUI may have the same strength as in species without DUI. Instead, it is tempting to speculate that generation of that kind of polymorphism in mtDNA depends on speciation/hybridization events, since they seem to coincide. Should that be so, the very existence of such rearranged haplotypes may indicate that the mechanisms of mitochondrial inheritance are not as different in animals with DUI as in animals with normal mtDNA inheritance: they both break at species boundaries.

ABBOTT, C. L., M. C. DOUBLE, J. W. H. TRUEMAN, A. ROBINSON and A. COCKBURN, 2005 An unusual source of apparent mitochondrial heteroplasmy: duplicate mitochondrial control regions in Thalassarche albatrosses. Mol. Ecol. 14: 3605–3613.[CrossRef][Medline]

BRETON, S., G. BURGER, D. T. STEWART and P. U. BLIER, 2006 Comparative analysis of gender-associated complete mitochondrial genomes in marine mussels (Mytilus spp.). Genetics 172: 1107–1119.[Abstract/Free Full Text]

BURZYSKI, A., M. ZBAWICKA, D. O. F. SKIBINSKI and R. WENNE, 2003 Evidence for Recombination of mtDNA in the marine mussel Mytilus trossulus from the Baltic. Mol. Biol. Evol. 20: 388–392.[Abstract/Free Full Text]

BURZYSKI, A., M. ZBAWICKA, D. O. F. SKIBINSKI and R. WENNE, 2006 Doubly uniparental inheritance is associated with high polymorphism for rearranged and recombinant control region haplotypes in Baltic Mytilus trossulus. Genetics 174: 1081–1094.[Abstract/Free Full Text]

CAO, L., E. KENCHINGTON, E. ZOUROS and G. C. RODAKIS, 2004 Evidence that the large noncoding sequence is the main control region of maternally and paternally transmitted mitochondrial genomes of the marine mussel (Mytilus spp.). Genetics 167: 835–850.[Abstract/Free Full Text]

EBERHARD, J. R., T. F. WRIGHT and 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]

GACH, M. H., and W. M. BROWN, 1997 Characteristics and distribution of large tandem duplications in brook stickleback (Culaea inconstans) mitochondrial DNA. Genetics 145: 383–394.[Abstract]

GJETVAJ, B., D. I. COOK and E. ZOUROS, 1992 Repeated sequences and large-scale size variation of mitochondrial DNA: a common feature among scallops (Bivalvia: Pectinidae). Mol. Biol. Evol. 9: 106–124.

KUMAZAWA, Y., H. OTA, M. NISHIDA and T. OZAWA, 1996 Gene rearrangements in snake mitochondrial genomes: highly concerted evolution of control-region-like sequences duplicated and inserted into a tRNA cluster. Mol. Biol. Evol. 13: 1242–1254.[Abstract]

LAVROV, D. V., J. L. BOORE and W. M. BROWN, 2002 Complete mtDNA sequences of two millipedes suggest a new model for mitochondrial gene rearrangements: duplication and nonrandom loss. Mol. Biol. Evol. 19: 163–169.[Abstract/Free Full Text]

MIZI, A., E. ZOUROS, N. MOSCHONAS and G. C. RODAKIS, 2005 The complete maternal and paternal mitochondrial genomes of the Mediterranean mussel Mytilus galloprovincialis: implications for the doubly uniparental inheritance mode of mtDNA. Mol. Biol. Evol. 22: 952–967.[Abstract/Free Full Text]

MIZI, A., E. ZOUROS and G. C. RODAKIS, 2006 Multiple events are responsible for an insertion in a paternally inherited mitochondrial genome of the mussel Mytilus galloprovincialis. Genetics 172: 2695–2698.[Abstract/Free Full Text]

MORITZ, C., and W. M. BROWN, 1987 Tandem duplications in animal mitochondrial DNAs: variation in incidence and gene content among lizards. Proc. Natl. Acad. Sci. USA 84: 7183–7187.[Abstract/Free Full Text]

RAWSON, P. D., and T. J. HILBISH, 1995 Evolutionary relationships among male and female mitochondrial DNA lineages in the Mytilus edulis species complex. Mol. Biol. Evol. 12: 893–901.[Abstract]

RIGINOS, C., M. J. HICKERSON, C. M. HENZLER and C. W. CUNNINGHAM, 2004 Differential patterns of male and female mtDNA exchange across the Atlantic Ocean in the blue mussel, Mytilus edulis. Evolution 58: 2438–2451.[CrossRef][Medline]

SKIBINSKI, D. O. F., C. GALLAGHER and C. M. BEYNON, 1994a Mitochondrial DNA inheritance. Nature 368: 817–818.

SKIBINSKI, D. O. F., C. GALLAGHER and C. M. BEYNON, 1994b Sex-limited mitochondrial DNA transmission in the marine mussel Mytilus edulis. Genetics 138: 801–809.[Abstract]

MIETANKA, B., M. ZBAWICKA, M. WOOWICZ and R. WENNE, 2004 Mitochondrial DNA lineages in the European populations of mussels Mytilus. Mar. Biol. 146: 79–92.[CrossRef]

STEWART, D. T., C. SAAVEDRA, R. STANWOOD, A. O. BALL and E. ZOUROS, 1995 Male and female mitochondrial DNA lineages in the blue mussel (Mytilus edulis) species group. Mol. Biol. Evol. 12: 735–747.[Abstract]

WENNE, R., and D. O. F. SKIBINSKI, 1995 Mitochondrial DNA heteroplasmy in European populations of mussel Mytilus trossulus. Mar. Biol. 122: 619–625.[CrossRef]

ZOUROS, E., A.O. BALL, C. SAAVEDRA and K. R. FREEMAN, 1994a Mitochondrial DNA inheritance. Nature 368: 818.[CrossRef][Medline]

ZOUROS, E., A. O. BALL, C. SAAVEDRA and K. R. FREEMAN, 1994b An unusual type of mitochondrial DNA inheritance in the blue mussel Mytilus. Proc. Natl. Acad. Sci. USA 91: 7463–7467.[Abstract/Free Full Text]

Communicating editor: M. J. SIMMONS

Online ISS 1091-6490
Copyright 2008 by the Genetics Society of America
phone: 412-268-1812   fax: 412-268-1813   email: genetics-gsa{at}andrew.cmu.edu