Scholarly article on topic 'Mitochondrial genomes and Doubly Uniparental Inheritance: new insights from Musculista senhousia sex-linked mitochondrial DNAs (Bivalvia Mytilidae)'

Mitochondrial genomes and Doubly Uniparental Inheritance: new insights from Musculista senhousia sex-linked mitochondrial DNAs (Bivalvia Mytilidae) Academic research paper on "Biological sciences"

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Academic research paper on topic "Mitochondrial genomes and Doubly Uniparental Inheritance: new insights from Musculista senhousia sex-linked mitochondrial DNAs (Bivalvia Mytilidae)"



Mitochondrial genomes and Doubly Uniparental Inheritance: new insights from Musculista senhousia sex-linked mitochondrial DNAs (Bivalvia Mytilidae)

Marco Passamonti*, Andrea Ricci, Liliana Milani and Fabrizio Ghiselli


Background: Doubly Uniparental Inheritance (DUI) is a fascinating exception to matrilinear inheritance of mitochondrial DNA (mtDNA). Species with DUI are characterized by two distinct mtDNAs that are inherited either through females (F-mtDNA) or through males (M-mtDNA). DUI sex-linked mitochondrial genomes share several unusual features, such as additional protein coding genes and unusual gene duplications/structures, which have been related to the functionality of DUI. Recently, new evidence for DUI was found in the mytilid bivalve Musculista senhousia. This paper describes the complete sex-linked mitochondrial genomes of this species.

Results: Our analysis highlights that both M and F mtDNAs share roughly the same gene content and order, but with some remarkable differences. The Musculista sex-linked mtDNAs have differently organized putative control regions (CR), which include repeats and palindromic motifs, thought to provide sites for DNA-binding proteins involved in the transcriptional machinery. Moreover, in male mtDNA, two cox2 genes were found, one (M-cox2b) 123bp longer.

Conclusions: The complete mtDNA genome characterization of DUI bivalves is the first step to unravel the complex genetic signals allowing Doubly Uniparental Inheritance, and the evolutionary implications of such an unusual transmission route in mitochondrial genome evolution in Bivalvia. The observed redundancy of the palindromic motifs in Musculista M-mtDNA may have a role on the process by which sperm mtDNA becomes dominant or exclusive of the male germline of DUI species. Moreover, the duplicated M-COX2b gene may have a different, still unknown, function related to DUI, in accordance to what has been already proposed for other DUI species in which a similar cox2 extension has been hypothesized to be a tag for male mitochondria.


Metazoan mitochondrial DNA (mtDNA) is generally a small molecule (15-20 kb), and although much larger mitochondrial genomes have occasionally been found, they are often products of duplications of mtDNA portions, rather than variations in gene content [1,2]. The typical mitochondrial gene complement encodes 13 protein subunits of the oxidative phosphorylation enzymes, 2 rRNAs and 22 tRNAs. However, the coding sequences (CDS) can be up to 16, the tRNAs up to 27 (source

* Correspondence:

Department of Biologia Evoluzionistica Sperimentale, University of Bologna, Bologna, Italy

MitoZoa: see [3]), and the rRNAs can be duplicated and/or fragmented in discontinuous genes, as in oysters [4]. Generally, there is also a single large non-coding region that is known to contain regulatory elements for replication and transcription (i.e. 'Control Region', CR), but it is unclear whether it is homologous among distantly related animals or, alternatively, it independently arose from various non-coding sequences. This difficulty in establishing homology is because CRs share sequence similarity only among closely related taxa. Finally, the mtDNA is almost always a circular molecule: only the cnidarian classes Cubozoa, Scyphozoa and Hydrozoa have been found to have linear mtDNA chromosomes [5]. All metazoan

O© 2011 Passamonti et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative CGntfäl Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

mitochondrial genes have homologs in plants, fungi and/or protists [6-9].

The Mollusca is the second largest animal Phylum and currently 99 complete mitochondrial genomes are available in Genbank; among those, only 38 are from Bival-via, the second class in terms of species richness among mollusks. So far, bivalve mtDNA displays an extraordinary amount of variation in gene arrangement, i.e. very few shared gene boundaries are detectable, and gene translocations are common across all gene classes (protein-coding genes, tRNAs and rRNAs). For this reason, bivalve mitochondrial genome may provide an excellent experimental system to review and test models of mt gene rearrangement evolution, which were mainly developed in groups with stable genomes, such as vertebrates or arthropods. In addition, gene duplications and/or losses are present in almost every bivalve taxon in which a complete mitochondrial genome is available (see [10]). It is therefore evident that efforts should be made to improve the knowledge of bivalve mitochon-drial genomes.

Another interesting feature of bivalve mtDNA is its unusual transmission route, which is found in some species: while in Metazoa mtDNA is known to be usually transmitted by Strict Maternal Inheritance (SMI; [11,12]), some bivalve mollusks show a deviation from this rule, named Doubly Uniparental Inheritance (DUI; [13,14]). DUI was found in species belonging to seven different bivalve families: Donacidae, Hyriidae, Margari-tiferidae, Mytilidae, Solenidae, Unionidae, and Veneridae ([15,16]). Species with DUI are characterized by the presence of two distinct gender-associated mtDNAs: one transmitted through eggs (F) and one transmitted through sperm (M). The F and M genomes show up to 52% nucleotide divergence [17]. DUI seems at first to violate the universal rule of uniparental inheritance of organelles, because males receive their mtDNA from both parents and their tissues are heteroplasmic. However the two mtDNAs segregate independently: the F-type is transmitted to the next generation only through females, while the M-type is only transmitted from father to sons, therefore both genomes are actually transmitted uniparentally.

Because of its unique features, DUI should be a choice model to address many aspects of a wide range of biological sub-fields such as mitochondria inheritance, mtDNA evolution and recombination, genomic conflicts, evolution of sex and developmental biology (see [18] for a review).

Recently, evidence for a new example of DUI was found in the mytilid Musculista senhousia [19]. In this work we characterized the two sex-linked mitochondrial genomes of M. senhousia, a step forward to the complete genetic characterization of DUI related sex-linked

mitochondrial genomes. In fact, several unusual features are coming to light when analyzing mtDNAs in DUI systems, such as additional protein coding genes ([20], and references therein) and gene duplications/features [21,22]. Functional explanations for these features will require much additional work, but are needed to understand the evolution and maintenance of DUI.


Mitochondrial genome features in M. senhousia

The obtained M. senhousia mtDNAs are 21,557 bp long in female (F-type) and 20,612 bp in male (M-type) (see Tables 1 and 2). Sequences are available in GenBank (Acc. No. GU001953-GU001954). The size of both F and M mitochondrial genomes are within the size range of mollusk mtDNAs sequenced to date, i.e. from 7808 bp in Batilaria cumingi to 32,115 bp in Placopecten magellanicus (source MitoZoa:; [3]).

M. senhousia F and M gene arrangements are remarkably different from other fully sequenced metazoan mtDNAs (see [10] for a review). Genome annotations are reported in Figure 1 and 2, Table 1 and 2. When compared to other Mytilidae, only four gene boundaries are shared with Mytilus (tRNAs are not considered), i.e. rrnS-nad6, nad2-cox3, nad4L-nad5 and nad3-cox1, while the rest of the genome is different, thus highlighting that gene arrangement evolves rapidly within the family.

Comparing the two sex linked genomes, protein-coding genes may have different lengths (Table 3). Both F-type and M-type include a large number of Unassigned Regions (URs; 29 in F and 27 in M: see Tables 1, 2 and Additional File 1). Among these, the largest (4,521 and 2,844 bp in female and male respectively) are here referred as LURs (i.e. Large Unassigned Regions).

Both F and M mt genomes show the same gene order and contain the full gene complement of the typical metazoan mtDNA, with two additional tRNAs: trnM and trnL (Figures 1 and 2; Tables 1 and 2). In males the cox2 gene is duplicated (Figure 2 and Table 2).

The atp8 gene was reported as missing in several bivalve mollusks, however, as recently reported [23], the lack of atp8 would rather be an annotation inaccuracy due to the extreme variability of the gene. Following [23], we found an atp8 gene in M. senhousia in both M and F genomes.

The position of the two ribosomal RNA genes, obtained through BLAST comparison, does not differ between male and female. In both sexes, rrnL is located in a region flanked by the trnM(AUG) and nad3 genes. Assuming that the first base at the 5'-end comes immediately after the trnM(AUG), and the 3'-end of the gene corresponds to the first base upstream of the start

Table 1 Organization of female Musculista senhousia mitochondrial genome.

Type Name Starts Stops Length Strand Anticodon Start Codon Stop Codon

GENE nad3 ' 390 390 H ATG TAA

UR UR-' 39' 625 235

tRNA trnY 626 69' 66 H GTA

UR UR-2 692 '234 543

tRNA trnH '235 '299 65 H GTG

UR UR-3 '300 '3'5 '6

tRNA trnI '316 '38' 66 H GAT

UR UR-4 '382 '39' '0

tRNA trnN '392 '457 66 H GTT

UR UR-5 '458 '564 '07

tRNA trnE '565 '63' 67 H TTC

LUR LUR '632 6'52 452'

GENE cox' 6'53 7736 '584 H ATG TAA

UR UR-6 7737 8"4 378

GENE cox2 8"5 8774 660 H ATA TAA

UR UR-7 8775 8832 58

GENE atp8 8833 8967 '35 H ATG TAA

UR UR-8 8968 905' 84 H

GENE atp6 9052 9765 7'4 H ATG TAG

UR UR-9 9766 979' 26

tRNA trnT 9792 9858 67 H TGT

GENE cob 9835 "03' "97 H ATA TAA

UR UR-'0 "032 "049 '8

tRNA trnD "050 ""4 65 H GTC

UR UR-'' ""5 '"23 9

tRNA trnR '"24 8 66 H TCG

tRNA trnS(AGN) '''9' "248 58 H TCT

UR UR-12 "249 "268 20

tRNA trnG "269 "336 68 H TCC

rRNA rrnS "337 ^54 8'8 H

GENE nad6 '2'55 '2778 624 H ATG TAA

UR UR-13 '2779 '2828 50

GENE nad2 '2829 '3773 945 H ATA TAA

UR UR-14 '3774 '3855 82

GENE cox3 '3856 855 H ATG TAA

UR UR-15 '47'' '472' ''

tRNA trnK '4722 '4792 7' H TTT

UR UR-16 '4793 '4797 5

tRNA trnF '4798 '4865 68 H GAA

UR UR-17 '4866 '4878 '3

tRNA trnP '4879 '4945 67 H TGG

UR UR-18 '4946 '4977 32

tRNA trnL(CUN) '4978 '5042 65 H TAG

UR UR-19 '5043 '5047 5

tRNA trnC '5048 '5"4 67 H GCA

UR UR-20 '5"5 '5'59 45

tRNA trnL(UUR) ^60 '5223 64 H TAA

UR UR-2' '5224 '5259 36

GENE nad' '5260 '6252 993 H ATG TAA

UR UR-22 '6253 '6385 '33

tRNA trnM(AUA) '6386 '6448 63 H TAT

Table 1 Organization of female Musculista senhousia mitochondrial genome. (Continued)

UR UR-23 16449 16486 38

tRNA trnV 16487 16550 64 H TAC

UR UR-24 1655' 16695 145

GENE nad4L 16696 16911 216 H ATA TAA

UR UR-25 16912 16988 77

GENE nad5 16989 18738 1750 H ATA T-

tRNA trnA 18739 18804 66 H TGC

UR UR-26 18805 18843 39

GENE nad4 18844 20163 1320 H ATA TAG

UR UR-27 20164 20213 50

tRNA trnW 20214 20280 67 H TCA

UR UR-28 2028- 20285 5

tRNA trnQ 20286 20353 68 H TTG

UR UR-29 20354 20360 7

tRNA trnM(AUG) 20361 20427 67 H CAT

rRNA rrnL 20428 21557 1130 H

codon of nad3 gene, the length of the rrnL genes are remarkably different: the male rrnL (1,682 bp in length) is 552 bp longer than the female one (1,130 bp in length). The rrnS gene is located in a region flanked by trnS and nad6 genes and, as above, we assumed that the first base at the 5'-end comes immediately after trnG, and that the 3'-end of the gene corresponds to the first base upstream of the start codon of nad6 gene. Here, the difference in length is reduced to 82 bp: the female rrnS gene is 819 bp long while the male one is 1,087 bp.

F and M genomes of M. senhousia contain 22 tRNA genes (see Tables 1, 2 and Additional File 2). As observed in mtDNA of some other mollusks (Katharina tunicata, Cepaea nemoralis, Mytilus species complex and Argopecten irradians), two leucine tRNA genes are present in M. senhousia. These can be differentiated by their anticodons: TAA for trnL(UUR) and TAG for trnL (CUN), which are 2-fold and 4-fold redundant respectively. Consequently, tnrL is 6-fold redundant. An additional trnM was also detected, as in V. philippinarum, Mytilus species complex, Crassostrea gigas, C. hongkon-gensis and C. virginica. The additional tRNA coding for methionine, trnM(AUA), has the TAT anticodon.

In both male and female mtDNAs, trnS(AGN) have a shortened DHU (See Additional File 2) that is not atypical, as this arm is unpaired in many metazoan taxa [24-27]. Moreover, mispairing between bases in stems is consistent across several taxa. For example, the second base pair in the anticodon stem of trn W has a T-T mis-pairing in Lampsilis ornata, Mytilus, and K. tunicata and a T-G pairing in several gastropods [25].

In the F mitochondrial genome of Musculista, 20 out of 22 tRNA genes are clustered in five groups of two to six (see Figure 1 and Table 1). Of the remaining two, trnT lies between atp6 and the 5'-end of cob genes

(with 24 bp overlapping each other) while trnA lies between nadS and nad4 genes. Thus, 4 of the 13 protein-coding genes (cob, nadl, nad4L and nad4) have a tRNA preceding their 5'-end. In contrast, 7 other genes (coxl, cox2, atp8, atp6, nad2, cox3 and nadS) have a non-coding sequence at their 5'-end that is capable of forming a stem and loop structure (see Figure 3).

In male mitochondrial DNA, 19 of the 22 tRNA genes are clustered in five groups ranging from two to six (see Figure 2 and Table 2). Of the remaining three, trnT lies between atp6 and the 5'-end of cob genes (with 25 bp overlapping each other), trnA lies between nadS and nad4 genes and trnE lies between the large unassigned region (LUR) and the 5'-end of coxl gene. Thus, 5 of the 14 protein-coding genes (coxl, cob, nadl, nad4L and nad4) have a tRNA preceding their 5'-end, while 7 other genes (cox2b, cox2, atp8, atp6, nad2, cox3 and nadS) have a non-coding sequence preceding their 5'-end that is capable of forming a stem and loop structure (see Figure 3). In a few cases those structures contain the translation initiation codon (coxl and cox2 in females, nad2 in males).

The nucleotide compositions of the two genomes are summarized in Table 3. Given the G content of the F and M coding strand (see Table 3), this can be considered as the heavy (H) strand of the molecule. The A+T content of the H strand is also high (66.5%, F; 67.0%, M). Variable values of A+T content are common in mollusks, and they have been reported in L. ornata (62%, [28]), Pupa strigosa (61.1%, [29]), and C. nemoralis (59.8%, [25]). In other mollusks, the A+T content is much higher (Albinaria coerulea, 70.7%, [30]; K. tunicata, 69.0%, [6]; Graptame eborea, 74.1%, [31]). Musculista values in A+T content are among the highest observed in the Phylum, and reflect the high

Table 2 Organization of male Musculista senhousia mitochondrial genome.

Type Name Starts Stops Length Strand Anticodon Start Codon Stop Codon

GENE nad3 ' 375 375 H ATG TAA

UR UR-' 376 433 58

tRNA trnY 434 50' 68 H GTA

UR UR-2 502 533 32

tRNA trnH 534 599 66 H GTG

UR UR-3 600 6'8 '9

tRNA trnI 6'9 688 70 H GAT

tRNA trnN 687 753 67 H GTT

LUR LUR 754 3597 2844

tRNA trnE 3598 3668 7' H TTC

UR UR-4 3669 3708 40

GENE cox' 3709 5292 '584 H ATG TAA

UR UR-5 5293 5852 560

GENE cox2b 5853 6665 8'3 H ATG TAA

UR UR-6 6666 6706 4'

GENE cox2 6707 7396 690 H ATA TAA

UR UR-7 7397 7402 6

GENE atp8 7403 7594 '92 H ATG TAG

UR UR-8 7595 76'2 '8

GENE atp6 76'3 8326 7'4 H ATG TAA

UR UR-9 8327 8347 2'

tRNA trnT 8348 84'6 69 H TGT

GENE cob 8392 9588 "97 H ATA TAA

UR UR-'0 9589 9606 '8

tRNA trnD 9607 967' 65 H GTC

UR UR-'' 9672 968' '0

tRNA trnR 9682 9745 64 H TCG

tRNA trnS(AGN) 9747 9806 60 H TCT

UR UR-'2 9807 9825 '9

tRNA trnG 9826 9893 68 H TCC

rRNA rrnS 9894 '0793 900 H

GENE nad6 '0794 "4'7 624 H ATG TAA

UR UR-n "4'8 "472 55

GENE nad2 "473 '24'7 945 H ATA TAG

UR UR-'4 '24'8 '2444 27

GENE cox3 '2445 '3299 855 H ATG TAG

tRNA trnK '3299 '3366 68 H TTT

UR UR-'5 '3367 '3377 ''

tRNA trnF '3378 '3445 68 H GAA

UR UR-'6 '3446 '3464 '9

tRNA trnP '3465 '3528 64 H TGG

UR UR-'7 '3529 '3554 26

tRNA trnL(CUN) '3555 '362' 67 H TAG

UR UR-'8 '3622 '3625 4

tRNA trnC '3626 '3696 7' H GCA

UR UR-'9 '3697 '3737 4'

tRNA trnL(UUR) '3738 '3804 67 H TAA

UR UR-20 '3805 '3840 36

GENE nad' '384' '4836 996 H ATG TAG

tRNA trnM(AUA) '4835 '4899 65 H TAT

UR UR-2' '4900 '4985 86

Table 2 Organization of male Musculista senhousia mitochondrial genome. (Continued)

tRNA trnV 14986 15049 64 H TAC

UR UR-22 15050 15183 134

GENE nad4L 15184 15399 216 H ATA TAA

UR UR-23 15400 15464 65

GENE nad5 15465 17229 1765 H ATA T-

tRNA trnA 17230 17294 65 H TGC

UR UR-24 17295 17338 44

GENE nad4 17339 18667 1329 H ATA TAA

UR UR-25 18668 18710 43

tRNA trnW 1871' 18777 67 H TCA

UR UR-26 18778 18781 4

tRNA trnQ 18782 18848 67 H TTG

UR UR-27 18849 18863 15

tRNA trnM(AUG) 18864 18930 67 H CAT

rRNA rrnL 18931 20612 1682 H

heterogeneity of molluscan mtDNA [2]. Moreover, there is a marked bias in favor of T against C, which is not restricted to any particular class of genes and does not differ between the two genomes.

The GC and AT asymmetry between the two mitochondrial DNA strands can be expressed in terms of GC skew and AT skew calculated according to [32]: GC skew = (G-C)/(G+C) and AT skew = (A-T)/(A+T), where G, C, A, and T are the occurrences of the four bases in the H strand. In M. senhousia F and M mito-chondrial genomes, the GC skew and the AT skew are F: +0.28 and -0.18, and M: +0.23 and -0.17, respectively.

In the M. senhousia male mtDNA 6 out of 14 protein genes start with the ATA codon and 8 with ATG, while in the female 7 out of 13 start with ATG and 6 with ATA (Tables 1 and 2). This pattern differs from that observed for Mytilus galloprovincialis, where 9 out of 13 protein genes start with the ATG codon, 2 with the ATA and 2 with GTG [23,33]. In all known metazoan mtDNAs, the most common start codon is ATG, and it is a general opinion that the methionine tRNA with the CAT anticodon represents the ancestral form. Moreover [24] suggested that the second methionine tRNA arose by duplication. The F and M genomes of the venerid Venerupis philippinarum also have two tRNA genes for methionine, but both have the ancestral CAT anticodon. TAA is the termination codon ten times in F and nine times in M mtDNA, while TAG is a stop codon two times in F, and four times in M. In both M and F genomes, nad5 gene is terminated by an incomplete termination codon T— (Tables 1 and 2), with their likely completion occurring by polyadenylation after transcript processing [34].

A total of 4,098 and 3,794 amino acids residues are encoded by male and female M. senhousia mitochon-drial genome respectively (Table 4). All codons do occur

in both Musculista mitochondrial genomes (Table 5). UUU (phenylalanine) is the most frequent codon, followed by UUA (leucine). UUU is also the most frequent codon in M. galloprovincialis [33], in L. ornata [28] and in C. nemoralis [35], whereas UUA (leucine) is most common in A. coerulea [30], P. strigosa [29], Roboastra europaea [36], G. eborea [31], and K. tunicata [6]. These two codons are also the most frequently used in other invertebrate mtDNAs [37-42]. UUU is also very frequent in basal chordates (e.g. amphioxus, Branchiostoma lan-ceolatum, [43]), but not in most vertebrates, where CUA (e.g., Cyprinus, [44]; Homo sapiens, [45]) or AUU (e.g., Xenopus laevis, [46]; Danio rerio, [47]) are the most frequent.

The least used codons in males are UCG (6), CCG (8) and CGG (8), while in females they are CCG (4), CGC (7) and UAG (7). Of these, CGC is also among the least common in the mtDNA of other mollusks. Synonymous codons, whether four-fold (4FD) or two-fold (2FD) degenerate, are recognized by the same tRNA, with the exception of the methionine codons, which are recognized by different tRNAs (Table 5).

Moreover, 2,754 F and 2,967 M Musculista codons (72.6% and 72.4% in female and in male respectively) end with an A or T, a more pronounced phenomenon than what observed for a typical invertebrate codon bias. There is a strong bias against the use of C (9.3% and 11.3% in female and in male respectively) at the third position nucleotide in all codons: in detail, for residues with a fourfold degenerate third position, codon families ending with T are the most frequently used (46.7% and 46.6% in female and male respectively). This is also the case for two-fold degenerate codons. In other words, in every case an amino acid residue can be specified by any NNY codon, both female and male M. senhousia mitochondrial genomes

Figure 1 Female Musculista senhousia mitochondrial genome. Gene map of the female Musculista senhousia mitochondrialgenome. Shortest URs (< 100 bp) are not indicated.

have a much higher proportion of NNT:NNC. In fact, female showed 44.7% of T and 9.3% of C, with NNT: NNC ratio of 4.8:1; while in male the ratio's value is slightly lower: 3.9:1 (43.8% of T and 11.2% of C). At the second position, there is even a stronger bias in favor of the use of T usage (45.4% and 44.2% in female and male respectively)(see Table 6), like in M. edulis (43.5%), C. hongkongensis (42.5%), C. gigas (42.3%) and C. virginica (43.0%).

Finally, in eight 2FD and seven 4FD codon families in females and in seven 2FD and seven 4FD codon families in males, the most frequently used codon does not match the tRNA anticodon. This has been observed in other metazoan mtDNA as well [46-50] and it suggests that strict codon-anticodon complementarity does not affect the codon composition of the genome. Deviations from equal frequency of the four nucleotides in 4FD sites are common in the animal mtDNA and have been attributed to several

Figure 2 Male Musculista senhousia mitochondrial genome. Gene map of the male Musculista senhousia mitochondrialgenome. Shortest URs (< 100 bp) are not indicated.

factors, such as unequal presence of the four nucleotides in the nucleotide pool, preference of the mitochondrial gamma DNA polymerase for specific nucleotides, or asymmetrical mutation rate owing to different duration of exposure of the lagging strand during replication [40,51-54].

Comparing the two M. senhousia sex linked genomes, the most conserved protein-coding genes are coxl and cob, and the least conserved are nad6 and atp8 (Table 4). Synonymous (Ks) and non-synonymous (Ka) substitution values between the two genomes do vary (Table 4). Ka is

particularly low for coxl (0.042), whereas Ks is not (0.838), suggesting that this gene is under some selective constraint (Ka/Ks = 0.05). The conservation of coxl is common in animal mtDNA [55,56]. In cob gene, both K values are lower than average (Table 4) with a Ka/Ks ratio's value (0.10) which is close to that of coxl gene.

The Large Unassigned Region (LUR)

As mentioned, in the female genome the LUR (F-LUR) is 4,521 bp long and it is included between trnE and the

Table 3 Length, base composition and sequence divergence of M, F genes and URs in Musculista senhousia.

Gene/Region F/M Length Base Composition (% T, C, A, G) pD ± SE

UR1-27/LUR M 4296 37.8 11.2 31.4 19.5 NA

UR1-29/LUR F 6798 37.9 10.4 30.8 20.9 NA

rrnL M 1682 37.3 12.6 30.8 19.3 0.343 ± 0.015

F 1130 35.8 13.5 30.4 20.3

rrnS M 900 36.0 11.6 33.1 19.3 0.093 ± 0.009

F 818 37.2 11.0 32.2 19.7

allrRNA genes M 2582 36.3 12.2 31.6 19.3 0.209 ± 0.010

F 1948 36.4 12.4 31.2 20.0

atp6 M 714 43.8 12.7 23.5 19.9 0.258 ± 0.016

F 714 42.2 12.9 23.8 21.1

atp8 M 192 42.2 14.1 27.6 16.1 0.281 ± 0.037

F 135 43.0 12.6 25.9 18.5

cox1 M 1584 38.3 15.9 24.7 21.1 0.180 ± 0.009

F 1584 40.0 14.4 24.4 21.3

cox2 M 690 36.7 15.2 26.7 21.4 0.264 ± 0.016

F 660 37.4 14.5 27.3 20.8

cox2b M 813 35.9 14.1 28.7 21.3 0.267 ± 0.016*


cox3 M 855 42.0 13.1 23.3 21.6 0.220 ± 0.012

F 855 43.4 12.9 20.9 22.8

cob M 1197 40.6 13.9 25.2 20.3 0.106 ± 0.009

F 1197 40.4 13.6 24.9 21.1

nad1 M 996 39.8 12.2 26.0 22.0 0.227 ± 0.014

F 993 41.3 11.5 24.4 23.2

nad2 M 945 44.9 10.8 24.4 19.9 0.302 ± 0.013

F 945 44.1 10.9 22.4 22.5

nad3 M 375 44.3 14.1 21.3 20.3 0.267 ± 0.021

F 390 45.6 12.6 21.0 20.8

nad4 M 1329 41.4 11.5 23.6 23.5 0.273 ± 0.013

F 1320 39.9 11.9 24.3 23.9

nad4L M 216 43.5 8.8 24.5 23.1 0.199 ± 0.027

F 216 44.0 8.8 24.5 22.7

nad5 M 1765 39.5 13.2 27.9 19.4 0.285 ± 0.011

F 1750 38.7 13.3 25.7 22.3

nad6 M 624 43.8 11.4 25.6 19.2 0.217 ± 0.017

F 624 42.1 12.3 25.2 20.4

allproteins M 12295 40.6 13.2 25.4 20.9 0.231 ± 0.004#

F 11383 40.9 12.8 24.1 22.1

complete M 20612 39.3 12.7 27.7 20.3 NA

F 21557 39.3 12.0 27.2 21.4

UR = Unassigned Regions. NA = Not Available. pD = p-Distance. SE = Standard Error.

*: pD between Mcox2 and Mcox2b genes.

#: Mcox2b gene was excluded from the computation of overall pD.

5'-end of coxl gene (Figure 1 and 4, Table 1), while in the male it (M-LUR) is 2,844 bp long, and included between trnN and trnE genes (Figure 2 and 4, Table 2). Both start with a dissimilar sequence/spacer 20 and 237 bp long, respectively.

The F-LUR contains two large repeats (Figure 4: Repl and Rep2) about 2,150 bp long (2,149 Repl; 2,151 Rep2), both subdividable in three regions: A, B and C (named A1, A2, B1, B2, C1 and C2; see Figure 4 and Additional File 3). Between Rep1 and Rep2, the A subregion is the most conserved (pD = 0.000, see Table 6) while C is the most variable, although with a low pD (0.010 ± 0.005). Overall, Rep1 and Rep2 have a pD of 0.004 ± 0.001. The region including the last 202 bp of the F-LUR shows some similarity (pD = 0.449 ± 0.035) to the A subregions (A1 and A2), for this reason it is indicated here as subregion A'.

All the A-type subregions (A1, A2 and A') start with a 46 bp conserved motif, named here a, that contains a 10 bp hairpin (ah; see Figure 5). Both the subunits C (C1 and C2) begin with a hairpin 27 bp long (Ch; Figure 5). The M-LUR contains an A-like subregion showing a pD of 0.362 ± 0.032 from A1 and A2 (Table 6), indicated as A'' (Figure 4). A'' starts with a 37 bp motif, here named a*, similar to a, but 9 bp shorter and with three mutations that allow the formation of a longer hairpin, here named a*h (31 bp; Figure 5), in comparison to the female hairpin ah. The M-LUR continues with the subunit B that is the most conserved region compared to the FLUR showing a pD from B1 and B2 of 0.098 ± 0.007 and 0.096 ± 0.007 respectively (Table 6). At the 3' end of B there is a motif, indicated as g (Figure 4) that is similar to the first part of the subunits C. g is repeated four times in tandem. The length of g1, g2 and g3 ranges from 268 and 265 bp while the last repeat, g4, is truncated and measures 17 bp (Additional File 3; Figure 4). The pD among the g motifs is low and ranges from 0.008 ± 0.005 in the female (between gc1 and gc2) and 0.019 ± 0.009 between g1 and g3 (Table 6). The pD of the g motifs between male and female varies from 0.346 and 0.350 ± 0.027 (Table 6). At the 5' end of each g motif a secondary structure is present (g1h, g2h, g3h and g4h respectively; Figure 5): g1h is 14 bp long, while the other three are 28 bp long. g2h and g3h are identical, g4h has a two bases mutation at the center of the loop and g1h is identical to the upper portion of g4h (see Figure 5).

Furthermore, in line with what has been found in other DUI bivalves, including Mytilus, an ORF coding for 121 amminoacids has been found in the F-LUR of M. senhousia. This protein was proposed to have a

Figure 3 Intergenic palindromes. Putative secondary structures preceding the 5'-end of some protein-coding genes. (F) Female Musculista senhousia mitochondrialgenome. (M) Male Musculista senhousia mitochondrialgenome.

functional role in DUI. Detailed analyses on this novel DUI related putative protein have been published in a more comparative way (see [20]).

The cox2 duplication in the male mtDNA

The male mtDNA contains an extra copy of the cox2 gene. This is not new for DUI animals, since the female mt genome of the marine clam V. philippinarum has a cox2 duplication as well (GenBank Acc. No. AB065375: Okazaki and Ueshima, unpublished).

In the female Musculista, the cox2 gene (Fcox2) is 660 bp long and is flanked by the "coxl/UR-6" and "UR-7/ atp8" regions at the 5'- and 3'-end respectively (see

Figure 1 and Table 1). In male mitochondrial genome, the two copies of cox2 are close to each other and linked by a little non coding region 41 bp long (UR-6). The two cox2 copies are located between "coxl/UR-5" and "UR-7/atp8" regions, and the first is 813 bp long, while the second is 690 bp long (Figure 2 and Table 2).

Bayesian phylogenetic analyses on Fcox2, Mcox2(690 bp), Mcox2(813 bp) genes and their homologous in Mytilus species, demonstrated that Fcox2 is more closely related to the shorter Mcox2 (690 bp), rather than to the longer one (Figure 6). For this reason, the 813 bp long Mcox2 seems to be an extra copy of the gene, and thus it is referred here as Mcox2b.

Table 4 Genes, gene lengths and divergences in male and female Musculista senhousia protein coding genes.

Protein gene Maa Faa pD ± SE Ks Ka Ka/Ks

atp6 238 238 0.228 ± 0.026 0.894 0. 56 0.17

atp8 64 45 0.302 ± 0.070 0.581 0.233 0.40

cox1 528 528 0.053 ± 0.009 0.838 0.042 0.05

cox2 230 220 0.25' ± 0.027 0.877 0. 78 0.20

cox2b* 27' NA 0.279 ± 0.029* 0.653* 0.223* 0.35*

cox3 285 285 0.155 ± 0.022 0.811 0. 07 0.13

cob 399 399 0.058 ± 0.012 0.346 0.034 0.10

nad1 332 331 0.218 ± 0.022 0.670 0.145 0.22

nad2 315 315 0.306 ± 0.026 0.843 0.244 0.29

nad3 125 130 0.218 ± 0.034 0.964 0. 62 0.17

nad4 443 440 0.243 ± 0.020 0.931 0. 75 0.19

nad4L 72 72 0.183 ± 0.045 0.626 0. 07 0.17

nad5 588 583 0.274 ± 0.018 0.862 0. 08 0.24

nad6 208 208 0.324 ± 0.031 0.619 0. 68 0.43

allproteins 4,098 3,794 0.716 0.143 0.20

Maa and Faa = number of amino acids in male and female respectively. pD = p-Distances at the amino acidic level.

Ks and Ka = divergence of protein genes in synonymous (Ks) and non synonymous (Ka) sites respectively. SE = Standard Error.

Ka/Ks = ratio values between Ka and Ks. *: comparisons between Mcox2 and Mcox2b genes.


Gene content and order of F and M Mitochondrial genomes in M. senhousia

In M. senhousia both M and F mtDNAs share the same gene content and order, except for a duplicated cox2 gene in males, and include the typical gene content of bivalve mtDNA. It has to be noted, however, that a common feature of bivalves is the apparent lack of the atp8 gene. For instance, [2] mentioned that a lack of the atp8 gene is one of several unusual features of the Myti-lus mt sequence. The atp8 gene was considered missing for almost all bivalve species studied so far, including Crassostrea hongkongensis, C. gigas, C. virginica, Placo-pecten magellanicus, Argopecten irradians, Mizuhopecten yessoensis and Acanthocardia tuberculata. On the contrary, the apt8 gene was found in Hiatella arctica, as well as in the female mitochondrial genome of the unionid bivalve L. ornata [28]. A remarkable observation is that V. philippinarum, another species with DUI [57], was recently found to contain a putative atp8 gene [58], which was not found in the first analyses; nonetheless, this gene apparently encodes 37 amino acids only and therefore has a questionable gene function. Finally, [23] examined ORFs from several bivalve mitochondrial genomes and found two novel ORFs (F-orf-ur4 and M-orf-ur4) in the largest unassigned region of F and M mytilid ones (UR-4: see [33]). BLASTN searches against EST_-others (all ESTs except human and mouse) showed that

both are transcribed in Mytilus spp. BLASTX and PSI-BLAST searches using inferred aminoacid sequences of F-orf-ur4 and M-orf-ur4 failed to detect any significant sequence similarity with known proteins, so the identity of those putative proteins is still unclear. Further analyses on structure and evolution patterns suggested that the novel ORFs "represent good candidates for the previously 'missing' atp8 in mytilid mtDNAs" [23]. Therefore, following [23], we also found atp8 putative genes in both sex-linked mitochondrial genomes of M. senhou-sia. Our atp8 genes share the same characteristics of the above mentioned proteins, so we are confident to annotate them as Musculista atp8 genes.

Generally speaking, most mtDNAs are characterized by strand asymmetry in term of gene distribution. In both M. senhousia mt genomes, all genes are transcribed from the same strand, i.e. the asymmetry is at its highest among Metazoa. Most marine bivalves also share this feature (Mytilus species-complex, C. gigas, C. virginica, C. hongkongensis and V. philippinarum). In contrast, this is not true for the two freshwater species L. ornata [28] and Inversidens japanensis (Acc. No. AB055625 and AB055624) (see also [59]). In other mollusks, a relatively small number of mitochondrial genes are transcribed from the second strand. The scaphopods G. eborea and S. lobatum are an exception, with about an equal number of genes encoded by each strand [31,58]. The occurrence of all genes in the same strand is a relatively rare phenomenon in metazoans and, in addition to bivalves, it has been reported in some annelids (Lumbricus terres-tris, [60]; Platynereis dumerilii, [61]) and brachiopods (Terebratulina retusa, [62]; Terebratalia transversa, [42]; Laqueus rubellus, [63]). Actually, almost 10% of the mitochondrial genomes examined to date do have all genes encoded in the same strand [10]. Moreover, most of the above mentioned groups, including Bivalvia, are also characterized by strong differences in gene content and/or gene order. This allowed [10] to suggest a possible correlation between these two features.

The trnS(AGN) could not be located with tRNAscan-SE [64] because of the absence of the DHU arm and therefore of a normal cloverleaf structure (see [27] for a detailed discussion), so we used the ARWEN software [65] to identify it. This unconventional tRNA was found also in several other animal groups ([27] and references therein), and it evolved very early in Metazoa [66]. In vitro analyses confirmed its functionality [67].

In Table 7, the distribution of trnS(UCN) and trnS (AGN) among bivalves is reported (only complete mitochondrial genomes included; source: mitozoa see [3]). Most of the species (22) have both the tRNAs, 7 only trnS(UCN) and 3 (including M. senhousia) only trnS(AGN). Placopecten magellanicus have two

Table 5 Codon usage in male and female Musculista senhousia mitochondrial genomes.


aa Codon Count % aa Codon Count % aa Codon Count % aa Codon Count % Codon Count %

Phe (F) UUU 303 8,0 Ser (S) UCU 107 2,8 Tyr(Y) UAU 125 3,3 Cys (C) UGU 80 2,1 UNU 615 16,2

UUC 36 0,9 UCC 8 0,2 UAC 39 1,0 UGC 14 0,4 UNC 97 2,6

Leu (L) UUA 254 6,7 UCA 36 0,9 s.c. (*) UAA 14 0,4 Trp (W) UGA 53 1,4 UNA 357 9,4

UUG 105 2,8 UCG 14 0,4 UAG 7 0,2 UGG 50 1,3 UNG 176 4,6

CUU 89 2,3 Pro (P) CCU 95 2,5 His (H) CAU 58 1,5 Arg (R) CGU 35 0,9 CNU 277 7,3

CUC 20 0,5 CCC 13 0,3 CAC 15 0,4 CGC 7 0,2 CNC 55 1,5

CUA 62 1,6 CCA 11 0,3 Glr (Q) CAA 32 0,8 CGA 14 0,4 CNA 119 3,1

CUG 4' 1,1 CCG 4 0,1 CAG 26 0,7 CGG 13 0,3 CNG 84 2,2

Ile (I) AUU 147 3,9 Thr (T) ACU 54 1,4 Asr (N) AAU 82 2,2 Ser (S) AGU 71 1,9 ANU 354 9,3

AUC 41 1,1 ACC 9 0,2 AAC 27 0,7 AGC 30 0,8 ANC 107 2,8

Met (M) AUA 139 3,7 ACA 29 0,8 Lys (K) AAA 81 2,1 AGA 90 2,4 ANA 339 8,9

AUG 62 1,6 ACG 17 0,4 AAG 33 0,9 AGG 68 1,8 ANG 180 4,7

Val(V) GUU 200 5,3 Ala (A) GCU 88 2,3 Asp (D) GAU 59 1,6 Gly (G) GGU 102 2,7 GNU 449 11,8

GUC 24 0,6 GCC 17 0,4 GAC 15 0,4 GGC 39 1,0 GNC 95 2,5

GUA 113 3,0 GCA 44 1,2 Glu (E) GAA 44 1,2 GGA 43 1,1 GNA 244 6,4

GUG 84 2,2 GCG 22 0,6 GAG 49 1,3 GGG 89 2,3 GNG 244 6,4

NUN 1720 45,4 NCN 568 15,0 NAN 706 18,6 NGN 798 21,0 Total 3792

aa Codor Court % aa Codor Court % aa Codor Court % aa Codor Court % Codor Court %

Phe (F) UUU 333 8,1 Ser (S) UCU 131 3,2 Tyr(Y) UAU 133 3,2 Cys (C) UGU 90 2,2 UNU 687 16,8

UUC 57 1,4 UCC 22 0,5 UAC 36 0,9 UGC 15 0,4 UNC 130 3,2

Leu (L) UUA 274 6,7 UCA 36 0,9 s.c. (*) UAA 18 0,4 Trp (W) UGA 69 1,7 UNA 397 9,7

UUG 104 2,5 UCG 6 0,1 UAG 10 0,2 UGG 46 1,1 UNG 166 4,1

CUU 86 2,1 Pro (P) CCU 91 2,2 His (H) CAU 51 1,2 Arg (R) CGU 42 1,0 CNU 270 6,6

CUC 16 0,4 CCC 14 0,3 CAC 30 0,7 CGC 11 0,3 CNC 71 1,7

CUA 55 1,3 CCA 20 0,5 Glr (Q) CAA 40 1,0 CGA 12 0,3 CNA 127 3,1

CUG 28 0,7 CCG 8 0,2 CAG 22 0,5 CGG 8 0,2 CNG 66 1,6

Ile (I) AUU 178 4,3 Thr (T) ACU 61 1,5 Asr (N) AAU 81 2,0 Ser (S) AGU 78 1,9 ANU 398 9,7

AUC 43 1,0 ACC 22 0,5 AAC 52 1,3 AGC 43 1,0 ANC 160 3,9

Met (M) AUA 148 3,6 ACA 35 0,9 Lys (K) AAA 104 2,5 AGA 97 2,4 ANA 384 9,4

AUG 79 1,9 ACG 12 0,3 AAG 38 0,9 AGG 75 1,8 ANG 204 5,0

Val(V) GUU 193 4,7 Ala (A) GCU 81 2,0 Asp (D) GAU 65 1,6 Gly (G) GGU 103 2,5 GNU 442 10,8

GUC 30 0,7 GCC 22 0,5 GAC 22 0,5 GGC 28 0,7 GNC 102 2,5

GUA 106 2,6 GCA 44 1,1 Glu (E) GAA 59 1,4 GGA 53 1,3 GNA 262 6,4

GUG 83 2,0 GCG 19 0,5 GAG 42 1,0 GGG 88 2,1 GNG 232 5,7

NUN 1813 44,2 NCN 624 15,2 NAN 803 19,6 NGN 858 20,9 Total 4098

Codons that match the corresponding tRNA anticodon are bold and underlined. aa: coded amminoacid. s.c.: stop codon.

copies of trnS(UCN), while Mizuhopecten yessoensis seems to lack a Serine tRNA. [68] suggested that the secondary structure of a tRNA gene between a pair of protein genes is responsible for the precise cleavage of the polycistronic primary transcript. In the absence of a tRNA, this role can be played by a stem-loop structure, the 5'-end part of the gene itself, or a combination of the two. Potential hairpin structures at protein-protein gene junctions with no intervening tRNA have been reported in several studies (e.g., [6,33,39,69,70]). Our

analysis demonstrated that putative hairpins are present in all the gene junctions in which a tRNA lacks, suggesting a functional role of such intergenic sequences (Figure 3).

The Large Unassigned Region (LUR) and the sex-linked mt-DNA transmission

The structure of the F and M LUR palindromes found are reported on Figure 4 and 5. The presence of palindromes within a mtDNA CR is not new; in fact, the

Table 6 p-Distance (± Standard Error) of LURs repeats, subregions and motifs.

Repl Rep2 0,004 0,001

Ai A2 0,000 0,000

Al/2 A'' 0,362 0,0 2

Al/2 A' 0,449 0,0 5

A'' A' 0,505 0,0

Bi B2 0,002 0,001

B2 B 0,096 0,007

Bi B 0,098 0,007

Cl C2 0,010 0,005

Yci ÏC2 0,008 0,005

Ï2 Уз 0,012 0,006

Ï2 g 0,015 0,007

Ï3 g 0,019 0,009

gCi/C2 g3 0, 46 0,027

gC1/C2 gl/2 0, 50 0,027

local fold symmetry created by the palindrome is thought to provide the site for DNA-binding proteins involved in the trascriptional machinery [71]. In more detail, palindromic motifs (and in general inverted repeats) have the potential to form single-stranded stem-loop cruciform structures which have been reported to be essential for replication of circular genomes in many prokaryotic and eukaryotic systems [72]. The redundancy of palindromic elements in the Musculista male LUR, when compared to that of the female, may be possibly related to an increased duplication ratio of the M mtDNA; we can also speculate that this feature may have some role in the process by which sperm mitochondrial DNA becomes dominant or exclusive of the male germline, although we know that this is also achieved through a differential segregation during early embryo development, and likely through a second, more strict, selection during primordial germ cells establishment (see [73]). Nevertheless, the question of how sperm mitochondrial DNA becomes dominant or the

exclusive component of the male germline in DUI species still remains open, and may be the outcome of various coordinated processes.

The duplication of the cox2 gene

One noteworthy finding of this analysis is the cox2 gene duplication in the male mtDNA, with the duplicated gene being longer than the original one, a feature that might be somehow related to DUI. In fact, an interesting analogy is evident with unionid bivalves, in which the male cox2 gene show a 200-codon extension, which is absent in the female mtDNA. Such a feature is found in all analyzed unionids so far, and it has been related to DUI functioning [21,22,74-76]. Actually, [21,22] proposed several hypotheses for the role the cox2 extension may have for DUI, but all are dependent upon identifying a specific function for it, which is not a trivial task. Moreover, they detected in the male gonad a poly-ade-nylated mRNA transcript of the cox2 gene that includes the extension, and they concluded that the extension is protein-coding and functional.

[21,22] also hypothesized that the COX2 protein extension might be involved in intracellular interactions determining the survival of the male mitochondrion. In other organisms, it has been shown that upon fertilization the sperm-derived mitochondria are targeted for elimination: a key process in sperm mitochondrial degradation is ubiquitination [77], in which mitochondria of paternal derivation are tagged with Ubiquitin and then degraded. In Mytilus, in which an Ubiquitin-like process has been proposed, this degradation would be sex-specific: the sperm-derived mitochondria survive in male embryos, whereas they are eliminated in females. All that considered, [21] proposed that the COX2 extension could be involved in blocking such elimination to ensure survival of the male mitochondrion, or, 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. An in situ hybridization seemed to demonstrate that the unionid male COX2 is present on both inner and outer membranes of the sperm mitochondria (see Figure 4 in [74]).

According to the above mentioned rationales, we hypothesize that the duplicated cox2b gene in male M. senhousia may represent a variant of what found in unionoidean bivalves, with proper signals for DUI mito-chondrial tagging lying in the COX2 protein extension of unionid bivalves, as well as in the duplicated COX2b protein of Musculista. A support to this view comes from the observation that an additional putative Trans Membrane Helix (TMH) is found in the 41 residue long tail of the Musculista COX2b, although this tail is considerably shorter that the unionid one (200 amminoa-cids). Actually, five putative TMHs were found in the unionid extended C-terminus of the male COX2, which led the Authors to hypothesize that it may have a functional significance for male unionoidean bivalve reproductive success [75,76].

In analogy, we suggest that COX2b might have some function related to mitochondrial tagging, like the COX2b and the Unionid COX2 extension. Further studies are needed to gain a more clear role of such proteins in the unusual DUI system of mitochondrial inheritance. Actually, a duplication similar to the Mus-culista one was also found in V. philippinarum, but quite surprisingly in the female mtDNA (see unpublished GenBank annotation). This suggests that cox2 duplication may be uncoupled with maleness. Moreover, no Mytilus genomes show a similar situation for cox2 or any other gene, so either duplicated genes or a cox2 tail may not be strictly necessary to sustain DUI.


The characteristics of the Musculista sex-linked mtDNAs evidently add to the knowledge of DUI systems, and highlight some unexpected features, shared among distantly related DUI species. Since it is commonly accepted that DUI is rather a variation of Strict

Figure 6 Bayesian tree for the cox2 genes. Cgi: Crassostrea gigas; Med: Mytilus edulis; Mga: Mytilus galloprovincialis; Mtr: Mytilus trossulus; Mse: Musculista senhousia.

Table 7 Serine tRNA [trnS(UCN) and trnS(AGN)] in bivalves.


Species (GenBank Acc. No.)




Mytiloida; Mytiloidea; Mytilidae



Ostreoida; Ostreoidea; Ostreidae

Pectinoida; Pectinoidea; Pectinidae

Musculista senhousia (GU001953) Mytilus edulis (AY823623) Mytilus galloprovincialis (AY363687) Mytilus trossulus (DQ198225)

Saccostrea mordax (FJ841968) Crassostrea angulata (FJ841965) Crassostrea ariakensis (FJ841964) Crassostrea gigas (NC_001276) Crassostrea hongkongensis (EU266073) Crassostrea iredalei (FJ841967) Crassostrea sikamea (FJ841966)

Mizuhopecten yessoensis (FJ595959) Chlamys farreri (EU715252) Mimachlamys nobilis (FJ595958) Placopecten magellanicus (NC_007234)* Argopecten irradians (NC_009687) Argopecten irradians irradians (DQ665851)

x x xx


Myoida; Hiatelloidea; Hiatellidae Veneroida; Cardioidea; Cardiidae Veneroida; Lucinoidea; Lucinidae

Veneroida; Tellinoidea; Solecurtidae Veneroida; Veneroidea; Veneridae

Hiatella arctica (NC_008451)

Acanthocardia tuberculata (NC_008452)

Loripes lacteus (EF043341) Lucinella divaricata (EF043342)

Sinonovacula constricta (EU880278)

Meretrix meretrix (GQ463598) Meretrix petechialis (EU145977) Venerupis philippinarum (AB065374) Paphia euglypta (GU269271)


Unionoida; Unionoidea; Unionidae

Ambleminae Anodontinae Anodontinae Unioninae

Venustaconcha ellipsiformis (FJ809752) Quadrula quadrula (FJ809750) Cristaria plicata (FJ986302) Pyganodon grandis (FJ809754) Hyriopsis cumingii (FJ529186) inversidens japanensis (AB055624) Unio pictorium (HM014131)

*: Placopecten magellanicus has two copies of trnS(UCN)

Note: only species with complete mitochondrial genomes available included.

Maternal Inheritance, than a completely different mechanism, we think that DUI is a good experimental model to better understand the general rules, as well as the molecular features of Metazoan mitochondrial inheritance (see [18], for a detailed discussion). For the above mentioned reasons, the complete mtDNA genome characterization of DUI bivalves is not only a mere descriptive exercise, but rather a first step to unravel the complex genetic signals allowing Doubly Uniparental Inheritance of mitochondrial DNA, and the evolutionary implications of such unusual transmission route in mitochondrial genome evolution in Bivalvia.


Sample Collection

Alive M. senhousia specimens from Venice Lagoon (Italy) were used for this analysis. Males and females were stimulated to spawn gametes in seawater supplemented with hydrogen peroxide, according to [78]. Each emission was analyzed with a light microscope to sex specimens. A total of 10 sperm and 10 egg samples were then collected after a gentle centrifugation (3,000 g). Seawater was removed, and ethanol added before storing samples at -20°C.

PCR analyses

Total genomic DNA was extracted using the DNeasy Tissue Kit (Qiagen), and partial sequences of cytochrome b (cob) and mitochondrial ribosomal large subunit RNA (rrnL) were amplified and directly sequenced (primers reported in Table 8), as described in [79]. Sequencing reactions were performed on both strands with BigDye Terminator Cycle Sequencing Kit according to supplier's instructions (Applied Biosystem) in a 310 Genetic Analyzer (ABI) automatic sequencer.

The 20 sequences obtained for both F and M genomes were aligned (not shown), and, after checking for variable sites, used to design sex-specific primers to amplify the entire mitochondrial genome in two overlapping fragments by long PCR reactions. LongPCR was performed on one Musculista specimen per sex. To

obtain the F genome, F-cob383R and F-16S142F

Table 8 Primer sequences.

Primer name Sequence









1 J.L. Boore, personal communication; 2 [88]

primers were used. The M genome was amplified with M-cob386R and M-16S103F. Both pairs of primers amplified a fragment of 10-11 kb respectively. Long PCR primer sequences are reported in Table 1. LongPCR amplifications were performed on a Gene Amp® PCR System 2720 (Applied Biosystem) in 50 ^l reaction volume composed of 31.5 ^l of sterilized distilled water, 10 ^l of 5 x Herculase II Fusion Reaction Buffer, 0.5 ^l of dNTPs mix (25 mM each dNTP), 1.25 ^l of each primer (10 ^M), 5 ^l of DNA template (2550 ng) and 0.5 ^l of Herculase II Fusion DNA Polymer-ase. Reaction conditions were according to supplier's recommendations: initial denaturation at 95°C for 5 min and then incubated at 95°C for 20 s, 50°C for 20 s, and 68°C for 10 min for 30 cycles and 68°C for 8 min for a final extension. Long-PCR fragments were then purified using Wizard® SV Gel and PCR Clean-Up System (Promega).

Shotgun cloning

Sequencing of the two LongPCR fragments was done using shotgun cloning: amplicons were randomly sheared to 1.2-1.5 kb DNA segments using a Hydro-Shear device (GeneMachines). Sheared DNA was blunt end repaired at room temperature for 60 min using 6 U of T4 DNA Polymerase (Roche), 30 U of DNA Polymerase I Klenow (NEB), 10 ^l of dNTPs mix, 13 ^l of 10 x NEB buffer 2 in a 115 ^l total volume, and then gel purified using the Wizard® SV Gel and PCR Clean-Up System (Promega). The resulting fragments were ligated into the SmaI site of a pUC18 cloning vector using the Fast-Link DNA ligation Kit (Epicentre) and electropo-rated into One Shot® T0P10 Electrocomp™ Escherichia coli cells (Invitrogen) using standard protocols. Clones were screened by PCR using M13 universal primers and recombinants were purified using Multiscreen (Milli-pore) according to the manufacturer's instructions. Clones were sequenced using M13 universal primers by Macrogen Inc. (Korea).

Raw sequences were manually corrected, and then assembled into contigs with Sequencher v.4.6 (Gene Codes). Hence, the final assemblies were based on a minimum sequence coverage of 3x.

Secondary structures and annotation

The tRNA genes were identified by their secondary structure using ARWEN [65], with invertebrate mito-chondrial codon predictors. Analysis of Open Reading Frames (ORFs) was performed with the ORF Finder program of NCBI gorf/ using the invertebrate mitochondrial genetic code. Sequences were identified using BLASTX, PSI-BLAST [80] and BLASTN [81] as implemented by the NCBI website

For all protein coding genes, alignments were computed with ClustalW [82].

When analyzing sequence variability, pairwise p-Dis-tances (pD), their mean values and standard errors (by the bootstrap procedure) were computed with MEGA v.5.03 [83]. In order to avoid any model of DNA substitution that can affect statistics (see [79]), the use of a pD was preferred.

The divergence of protein genes in synonymous (Ks) and non-synonymous (Ka) sites was calculated by the modified Nei-Gojobori method with the Jukes-Cantor correction; the pD at the residue level was also calculated within the MEGA v.5.03 environment [83].

Two-fold, and four-fold degenerated positions were identified using DnaSP v.5 [84]. The Sequence Manipulation Suite (; [85]) was used to estimate codon usage. Potential DNA secondary structures near or at the 5'-end of protein genes were predicted using the UNAFold software package [86] available on the DINAMelt web server (http://; [86]).

Bayesian analyses on cox2 genes was performed with the MrBayes 3.1 (5,000,000 generations; [87]).

Additional material

Additional file 1: The Unassigned Regions (URs) in the female and male mtDNAs of Musculista senhousia. Annotation and length of Unassigned Regions (URs) in the female (Mse_URs_F) and male (Mse_URs_M) mtDNAs of Musculista senhousia.

Additional file 2: tRNAs in the female and male mtDNAs of Musculista senhousia. Annotation, length and structures of tRNAs in the female (Mse_trn_F) and male (Mse_trn_M) mtDNAs of Musculista senhousia.

Additional file 3: Structure of the female (F-LUR) and male (M-LUR) Large Unassigned Regions of Musculista senhousia mtDNA.

Schematic table of repeats and hairpin structures in the Large Unassigned Regions (LURs) of the female and male Musculista senhousia mtDNAs (F-LUR and M-LUR).


We want to thank Edoardo Turolla (C.Ri.M., Goro, Italy) for providing us Musculista samples. This work was supported by the University and Research Italian Ministry (MIUR PRIN07, grant number 2007NSHJL8_002 to MP) and the "Canziani Bequest" fund (University of Bologna, grant number A.31. CANZELSEW to MP).

Authors' contributions

MP conceived the study, participated in its design and coordination and drafted the manuscript. AR carried out the lab work and performed part of the analysis. LM and FG performed part of the analysis and drafted the manuscript. Allauthors read and approved the finalmanuscript.

Received: 24 May 2011 Accepted: 6 September 2011 Published: 6 September 2011


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Cite this article as: Passamonti et al.: Mitochondrial genomes and Doubly Uniparental Inheritance: new insights from Musculista senhousia sex-linked mitochondrial DNAs (Bivalvia Mytilidae). BMC Genomics 2011 12:442.

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