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

  • Marco Passamonti1Email author,

    Affiliated with

    • Andrea Ricci1,

      Affiliated with

      • Liliana Milani1 and

        Affiliated with

        • Fabrizio Ghiselli1

          Affiliated with

          BMC Genomics201112:442

          DOI: 10.1186/1471-2164-12-442

          Received: 24 May 2011

          Accepted: 6 September 2011

          Published: 6 September 2011

          Abstract

          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.

          Background

          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 MitoZoa: http://​mi.​caspur.​it/​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 mitochondrial genes have homologs in plants, fungi and/or protists [69].

          The Mollusca is the second largest animal Phylum and currently 99 complete mitochondrial genomes are available in Genbank; among those, only 38 are from Bivalvia, 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 mitochondrial 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, Margaritiferidae, 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.

          Results

          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: http://​mi.​caspur.​it/​mitozoa; [3]).
          Table 1

          Organization of female Musculista senhousi a mitochondrial genome.

          Type

          Name

          Starts

          Stops

          Length

          Strand

          Anticodon

          Start Codon

          Stop Codon

          GENE

          nad3

          1

          390

          390

          H

           

          ATG

          TAA

          UR

          UR-1

          391

          625

          235

              

          tRNA

          trnY

          626

          691

          66

          H

          GTA

            

          UR

          UR-2

          692

          1234

          543

              

          tRNA

          trnH

          1235

          1299

          65

          H

          GTG

            

          UR

          UR-3

          1300

          1315

          16

              

          tRNA

          trnI

          1316

          1381

          66

          H

          GAT

            

          UR

          UR-4

          1382

          1391

          10

              

          tRNA

          trnN

          1392

          1457

          66

          H

          GTT

            

          UR

          UR-5

          1458

          1564

          107

              

          tRNA

          trnE

          1565

          1631

          67

          H

          TTC

            

          LUR

          LUR

          1632

          6152

          4521

              

          GENE

          cox1

          6153

          7736

          1584

          H

           

          ATG

          TAA

          UR

          UR-6

          7737

          8114

          378

              

          GENE

          cox2

          8115

          8774

          660

          H

           

          ATA

          TAA

          UR

          UR-7

          8775

          8832

          58

              

          GENE

          atp8

          8833

          8967

          135

          H

           

          ATG

          TAA

          UR

          UR-8

          8968

          9051

          84

          H

             

          GENE

          atp6

          9052

          9765

          714

          H

           

          ATG

          TAG

          UR

          UR-9

          9766

          9791

          26

              

          tRNA

          trnT

          9792

          9858

          67

          H

          TGT

            

          GENE

          cob

          9835

          11031

          1197

          H

           

          ATA

          TAA

          UR

          UR-10

          11032

          11049

          18

              

          tRNA

          trnD

          11050

          11114

          65

          H

          GTC

            

          UR

          UR-11

          11115

          11123

          9

              

          tRNA

          trnR

          11124

          11189

          66

          H

          TCG

            

          tRNA

          trnS(AGN)

          11191

          11248

          58

          H

          TCT

            

          UR

          UR-12

          11249

          11268

          20

              

          tRNA

          trnG

          11269

          11336

          68

          H

          TCC

            

          rRNA

          rrnS

          11337

          12154

          818

          H

             

          GENE

          nad6

          12155

          12778

          624

          H

           

          ATG

          TAA

          UR

          UR-13

          12779

          12828

          50

              

          GENE

          nad2

          12829

          13773

          945

          H

           

          ATA

          TAA

          UR

          UR-14

          13774

          13855

          82

              

          GENE

          cox3

          13856

          14710

          855

          H

           

          ATG

          TAA

          UR

          UR-15

          14711

          14721

          11

              

          tRNA

          trnK

          14722

          14792

          71

          H

          TTT

            

          UR

          UR-16

          14793

          14797

          5

              

          tRNA

          trnF

          14798

          14865

          68

          H

          GAA

            

          UR

          UR-17

          14866

          14878

          13

              

          tRNA

          trnP

          14879

          14945

          67

          H

          TGG

            

          UR

          UR-18

          14946

          14977

          32

              

          tRNA

          trnL(CUN)

          14978

          15042

          65

          H

          TAG

            

          UR

          UR-19

          15043

          15047

          5

              

          tRNA

          trnC

          15048

          15114

          67

          H

          GCA

            

          UR

          UR-20

          15115

          15159

          45

              

          tRNA

          trnL(UUR)

          15160

          15223

          64

          H

          TAA

            

          UR

          UR-21

          15224

          15259

          36

              

          GENE

          nad1

          15260

          16252

          993

          H

           

          ATG

          TAA

          UR

          UR-22

          16253

          16385

          133

              

          tRNA

          trnM(AUA)

          16386

          16448

          63

          H

          TAT

            

          UR

          UR-23

          16449

          16486

          38

              

          tRNA

          trnV

          16487

          16550

          64

          H

          TAC

            

          UR

          UR-24

          16551

          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

          20281

          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

             
          Table 2

          Organization of male Musculista senhousia mitochondrial genome.

          Type

          Name

          Starts

          Stops

          Length

          Strand

          Anticodon

          Start Codon

          Stop Codon

          GENE

          nad3

          1

          375

          375

          H

           

          ATG

          TAA

          UR

          UR-1

          376

          433

          58

              

          tRNA

          trnY

          434

          501

          68

          H

          GTA

            

          UR

          UR-2

          502

          533

          32

              

          tRNA

          trnH

          534

          599

          66

          H

          GTG

            

          UR

          UR-3

          600

          618

          19

              

          tRNA

          trnI

          619

          688

          70

          H

          GAT

            

          tRNA

          trnN

          687

          753

          67

          H

          GTT

            

          LUR

          LUR

          754

          3597

          2844

              

          tRNA

          trnE

          3598

          3668

          71

          H

          TTC

            

          UR

          UR-4

          3669

          3708

          40

              

          GENE

          cox1

          3709

          5292

          1584

          H

           

          ATG

          TAA

          UR

          UR-5

          5293

          5852

          560

              

          GENE

          cox2b

          5853

          6665

          813

          H

           

          ATG

          TAA

          UR

          UR-6

          6666

          6706

          41

              

          GENE

          cox2

          6707

          7396

          690

          H

           

          ATA

          TAA

          UR

          UR-7

          7397

          7402

          6

              

          GENE

          atp8

          7403

          7594

          192

          H

           

          ATG

          TAG

          UR

          UR-8

          7595

          7612

          18

              

          GENE

          atp6

          7613

          8326

          714

          H

           

          ATG

          TAA

          UR

          UR-9

          8327

          8347

          21

              

          tRNA

          trnT

          8348

          8416

          69

          H

          TGT

            

          GENE

          cob

          8392

          9588

          1197

          H

           

          ATA

          TAA

          UR

          UR-10

          9589

          9606

          18

              

          tRNA

          trnD

          9607

          9671

          65

          H

          GTC

            

          UR

          UR-11

          9672

          9681

          10

              

          tRNA

          trnR

          9682

          9745

          64

          H

          TCG

            

          tRNA

          trnS(AGN)

          9747

          9806

          60

          H

          TCT

            

          UR

          UR-12

          9807

          9825

          19

              

          tRNA

          trnG

          9826

          9893

          68

          H

          TCC

            

          rRNA

          rrnS

          9894

          10793

          900

          H

             

          GENE

          nad6

          10794

          11417

          624

          H

           

          ATG

          TAA

          UR

          UR-13

          11418

          11472

          55

              

          GENE

          nad2

          11473

          12417

          945

          H

           

          ATA

          TAG

          UR

          UR-14

          12418

          12444

          27

              

          GENE

          cox3

          12445

          13299

          855

          H

           

          ATG

          TAG

          tRNA

          trnK

          13299

          13366

          68

          H

          TTT

            

          UR

          UR-15

          13367

          13377

          11

              

          tRNA

          trnF

          13378

          13445

          68

          H

          GAA

            

          UR

          UR-16

          13446

          13464

          19

              

          tRNA

          trnP

          13465

          13528

          64

          H

          TGG

            

          UR

          UR-17

          13529

          13554

          26

              

          tRNA

          trnL(CUN)

          13555

          13621

          67

          H

          TAG

            

          UR

          UR-18

          13622

          13625

          4

              

          tRNA

          trnC

          13626

          13696

          71

          H

          GCA

            

          UR

          UR-19

          13697

          13737

          41

              

          tRNA

          trnL(UUR)

          13738

          13804

          67

          H

          TAA

            

          UR

          UR-20

          13805

          13840

          36

              

          GENE

          nad1

          13841

          14836

          996

          H

           

          ATG

          TAG

          tRNA

          trnM(AUA)

          14835

          14899

          65

          H

          TAT

            

          UR

          UR-21

          14900

          14985

          86

              

          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

          18711

          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

             
          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.
          http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-442/MediaObjects/12864_2011_3583_Fig1_HTML.jpg
          Figure 1

          FemaleMusculista senhousiamitochondrial genome. Gene map of the female Musculista senhousia mitochondrial genome. Shortest URs (< 100 bp) are not indicated.

          http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-442/MediaObjects/12864_2011_3583_Fig2_HTML.jpg
          Figure 2

          MaleMusculista senhousiamitochondrial genome. Gene map of the male Musculista senhousia mitochondrial genome. Shortest URs (< 100 bp) are not indicated.

          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).
          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

           

          all rRNA 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*

           

          F

          NA

          NA

           

          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

           

          all proteins

          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.

          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 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. hongkongensis 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 [2427]. Moreover, mispairing between bases in stems is consistent across several taxa. For example, the second base pair in the anticodon stem of trnW has a T-T mispairing in Lampsilis 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 nad5 and nad4 genes. Thus, 4 of the 13 protein-coding genes (cob, nad1, nad4L and nad4) have a tRNA preceding their 5'-end. In contrast, 7 other genes (cox1, cox2, atp8, atp6, nad2, cox3 and nad5) have a non-coding sequence at their 5'-end that is capable of forming a stem and loop structure (see Figure 3).
          http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-442/MediaObjects/12864_2011_3583_Fig3_HTML.jpg
          Figure 3

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

          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 nad5 and nad4 genes and trnE lies between the large unassigned region (LUR) and the 5'-end of cox1 gene. Thus, 5 of the 14 protein-coding genes (cox1, cob, nad1, nad4L and nad4) have a tRNA preceding their 5'-end, while 7 other genes (cox2b, cox2, atp8, atp6, nad2, cox3 and nad5) 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 (cox1 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 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 mitochondrial 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 mitochondrial 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 [3742]. UUU is also very frequent in basal chordates (e.g. amphioxus, Branchiostoma lanceolatum, [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.
          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

          0.156

          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

          0.251 ± 0.027

          0.877

          0.178

          0.20

          cox2b*

          271

          NA

          0.279 ± 0.029*

          0.653*

          0.223*

          0.35*

          cox3

          285

          285

          0.155 ± 0.022

          0.811

          0.107

          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

          0.162

          0.17

          nad4

          443

          440

          0.243 ± 0.020

          0.931

          0.175

          0.19

          nad4L

          72

          72

          0.183 ± 0.045

          0.626

          0.107

          0.17

          nad5

          588

          583

          0.274 ± 0.018

          0.862

          0.208

          0.24

          nad6

          208

          208

          0.324 ± 0.031

          0.619

          0.268

          0.43

          all proteins

          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.

          Table 5

          Codon usage in male and female Musculista senhousia mitochondrial genomes.

          FEMALE

          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

          Gln (Q)

          CAA

          32

          0,8

           

          CGA

          14

          0,4

          CNA

          119

          3,1

           

          CUG

          41

          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

          Asn (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

           

          MALE

          aa

          Codon

          Count

          %

          aa

          Codon

          Count

          %

          aa

          Codon

          Count

          %

          aa

          Codon

          Count

          %

          Codon

          Count

          %

          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

          Gln (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

          Asn (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.

          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 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%).
          Table 6

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

            

          pD

          SE

          Rep1

          Rep2

          0,004

          0,001

          A1

          A2

          0,000

          0,000

          A1/2

          A''

          0,362

          0,032

          A1/2

          A'

          0,449

          0,035

          A''

          A'

          0,505

          0,033

          B1

          B2

          0,002

          0,001

          B2

          B

          0,096

          0,007

          B1

          B

          0,098

          0,007

          C1

          C2

          0,010

          0,005

          γ C1

          γ C2

          0,008

          0,005

          γ 2

          γ 3

          0,012

          0,006

          γ 2

          γ 1

          0,015

          0,007

          γ 3

          γ 1

          0,019

          0,009

          γ C1/C2

          γ 3

          0,346

          0,027

          γ C1/C2

          γ 1/2

          0,350

          0,027

          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 [4650] 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 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, 5154].

          Comparing the two M. senhousia sex linked genomes, the most conserved protein-coding genes are cox1 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 cox1 (0.042), whereas Ks is not (0.838), suggesting that this gene is under some selective constraint (Ka/Ks = 0.05). The conservation of cox1 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 cox1 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 5'-end of cox1 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.
          http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-442/MediaObjects/12864_2011_3583_Fig4_HTML.jpg
          Figure 4

          Large Unassigned Regions (LURs). Schematic structure of female (F) and male (M) LURs in Musculista senhousia.

          The F-LUR contains two large repeats (Figure 4: Rep1 and Rep2) about 2,150 bp long (2,149 Rep1; 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 α, that contains a 10 bp hairpin (αh; 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 α*, similar to α, but 9 bp shorter and with three mutations that allow the formation of a longer hairpin, here named α*h (31 bp; Figure 5), in comparison to the female hairpin αh. The M-LUR continues with the subunit B that is the most conserved region compared to the F-LUR 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 γ (Figure 4) that is similar to the first part of the subunits C. γ is repeated four times in tandem. The length of γ1, γ2 and γ3 ranges from 268 and 265 bp while the last repeat, γ4, is truncated and measures 17 bp (Additional File 3; Figure 4). The pD among the γ motifs is low and ranges from 0.008 ± 0.005 in the female (between γc1 and γc2) and 0.019 ± 0.009 between γ1 and γ3 (Table 6). The pD of the γ motifs between male and female varies from 0.346 and 0.350 ± 0.027 (Table 6). At the 5' end of each γ motif a secondary structure is present (γ1h, γ2h, γ3h and γ4h respectively; Figure 5): γ1h is 14 bp long, while the other three are 28 bp long. γ2h and γ3h are identical, γ4h has a two bases mutation at the center of the loop and γ1h is identical to the upper portion of γ4h (see Figure 5).
          http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-442/MediaObjects/12864_2011_3583_Fig5_HTML.jpg
          Figure 5

          LUR palindromes. Sequences and structures of palindromic motifs located in the Musculista senhousia LURs.

          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 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 "cox1/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 "cox1/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.
          http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-442/MediaObjects/12864_2011_3583_Fig6_HTML.jpg
          Figure 6

          Bayesian tree for thecox2genes. Cgi: Crassostrea gigas; Med: Mytilus edulis; Mga: Mytilus galloprovincialis; Mtr: Mytilus trossulus; Mse: Musculista senhousia.

          Discussion

          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 Mytilus mt sequence. The atp8 gene was considered missing for almost all bivalve species studied so far, including Crassostrea hongkongensis, C. gigas, C. virginica, Placopecten 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. senhousia. 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 terrestris, [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: http://​mi.​caspur.​it/​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 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).
          Table 7

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

          Taxonomy

          Species (GenBank Acc. No.)

          Missing

          UCN

          AGN

          UCN+AGN

          Pteriomorphia

               

          Mytiloida; Mytiloidea; Mytilidae

               

          Crenellinae

          Musculista senhousia (GU001953)

            

          x

           

          Mytilinae

          Mytilus edulis (AY823623)

             

          x

           

          Mytilus galloprovincialis (AY363687)

             

          x

           

          Mytilus trossulus (DQ198225)

             

          x

          Ostreoida; Ostreoidea; Ostreidae

               
           

          Saccostrea mordax (FJ841968)

            

          x

           
           

          Crassostrea angulata (FJ841965)

             

          x

           

          Crassostrea ariakensis (FJ841964)

             

          x

           

          Crassostrea gigas (NC_001276)

             

          x

           

          Crassostrea hongkongensis (EU266073)

             

          x

           

          Crassostrea iredalei (FJ841967)

             

          x

           

          Crassostrea sikamea (FJ841966)

             

          x

          Pectinoida; Pectinoidea; Pectinidae

               
           

          Mizuhopecten yessoensis (FJ595959)

          x

             
           

          Chlamys farreri (EU715252)

           

          x

            
           

          Mimachlamys nobilis (FJ595958)

           

          x

            
           

          Placopecten magellanicus (NC_007234)*

           

          xx

            
           

          Argopecten irradians (NC_009687)

             

          x

           

          Argopecten irradians irradians (DQ665851)

            

          x

           

          Heteroconchia

               

          Myoida; Hiatelloidea; Hiatellidae

               
           

          Hiatella arctica (NC_008451)

             

          x

          Veneroida; Cardioidea; Cardiidae

               
           

          Acanthocardia tuberculata (NC_008452)

             

          x

          Veneroida; Lucinoidea; Lucinidae

               
           

          Loripes lacteus (EF043341)

             

          x

           

          Lucinella divaricata (EF043342)

             

          x

          Veneroida; Tellinoidea; Solecurtidae

               
           

          Sinonovacula constricta (EU880278)

           

          x

            

          Veneroida; Veneroidea; Veneridae

               
           

          Meretrix meretrix (GQ463598)

           

          x

            
           

          Meretrix petechialis (EU145977)

           

          x

            
           

          Venerupis philippinarum (AB065374)

           

          x

            
           

          Paphia euglypta (GU269271)

             

          x

          Palaeoheterodonta

               

          Unionoida; Unionoidea; Unionidae

               
           

          Venustaconcha ellipsiformis (FJ809752)

             

          x

          Ambleminae

          Quadrula quadrula (FJ809750)

             

          x

          Anodontinae

          Cristaria plicata (FJ986302)

             

          x

          Anodontinae

          Pyganodon grandis (FJ809754)

             

          x

          Unioninae

          Hyriopsis cumingii (FJ529186)

             

          x

           

          Inversidens japanensis (AB055624)

             

          x

           

          Unio pictorium (HM014131)

             

          x

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

          Note: only species with complete mitochondrial genomes available included.

          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 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, 7476]. 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-adenylated 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 mitochondrial 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 amminoacids). 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 Musculista 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.

          Conclusions

          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 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.

          Methods

          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.
          Table 8

          Primer sequences.

          Primer name

          Sequence

          cobR1

          5'-GCRTAWGCRAAWARRAARTAYCAYTCWGG-3'

          cobF1

          16Sbr2

          5'-GGWTAYGTWYTWCCWTGRGGWCARAT-3'

          5'-CCGGTCTGAACTCAGATCACGT-3'

          16Sar2

          5'-CGCCTGTTTATCAAAAACAT-3'

          F-cob383R

          F-16S142F

          5'-TAGGAGTTTTTATAGGGTCTGC-3'

          5'-ACCTGAAGTTGTCTCATTTACC-3'

          M-cob386R

          M-16S103F

          5'-GGATAGGAGTTTTTATAGGGTCTGC-3'

          5'-GTGAATTTCTTAGAGTGACGATTA-3'

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

          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 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 μl reaction volume composed of 31.5 μl of sterilized distilled water, 10 μl of 5 × Herculase II Fusion Reaction Buffer, 0.5 μl of dNTPs mix (25 mM each dNTP), 1.25 μl of each primer (10 μM), 5 μl of DNA template (25-50 ng) and 0.5 μl of Herculase II Fusion DNA Polymerase. 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 HydroShear 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 μl of dNTPs mix, 13 μl of 10 × NEB buffer 2 in a 115 μl 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 electroporated into One Shot® TOP10 Electrocomp™ Escherichia coli cells (Invitrogen) using standard protocols. Clones were screened by PCR using M13 universal primers and recombinants were purified using Multiscreen (Millipore) 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 3×.

          Secondary structures and annotation

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

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

          When analyzing sequence variability, pairwise p-Distances (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 (http://​www.​bioinformatics.​org/​sms2; [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://​mfold.​rna.​albany.​edu/​?​q=​DINAMelt; [86]).

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

          Declarations

          Acknowledgements

          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’ Affiliations

          (1)
          Department of Biologia Evoluzionistica Sperimentale, University of Bologna

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