A comparative study of nemertean complete mitochondrial genomes, including two new ones for Nectonemertes cf. mirabilis and Zygeupolia rubens, may elucidate the fundamental pattern for the phylum Nemertea

  • Hai-Xia Chen1, 2,

    Affiliated with

    • Shi-Chun Sun2,

      Affiliated with

      • Per Sundberg1,

        Affiliated with

        • Wei-Cheng Ren3 and

          Affiliated with

          • Jon L Norenburg4Email author

            Affiliated with

            BMC Genomics201213:139

            DOI: 10.1186/1471-2164-13-139

            Received: 2 May 2011

            Accepted: 17 April 2012

            Published: 17 April 2012

            Abstract

            Background

            The mitochondrial genome is important for studying genome evolution as well as reconstructing the phylogeny of organisms. Complete mitochondrial genome sequences have been reported for more than 2200 metazoans, mainly vertebrates and arthropods. To date, from a total of about 1275 described nemertean species, only three complete and two partial mitochondrial DNA sequences from nemerteans have been published. Here, we report the entire mitochondrial genomes for two more nemertean species: Nectonemertes cf. mirabilis and Zygeupolia rubens.

            Results

            The sizes of the entire mitochondrial genomes are 15365 bp for N. cf. mirabilis and 15513 bp for Z. rubens. Each circular genome contains 37 genes and an AT-rich non-coding region, and overall nucleotide composition is AT-rich. In both species, there is significant strand asymmetry in the distribution of nucleotides, with the coding strand being richer in T than A and in G than C. The AT-rich non-coding regions of the two genomes have some repeat sequences and stem-loop structures, both of which may be associated with the initiation of replication or transcription. The 22 tRNAs show variable substitution patterns in nemerteans, with higher sequence conservation in genes located on the H strand. Gene arrangement of N. cf. mirabilis is identical to that of Paranemertes cf. peregrina, both of which are Hoplonemertea, while that of Z. rubens is the same as in Lineus viridis, both of which are Heteronemertea. Comparison of the gene arrangements and phylogenomic analysis based on concatenated nucleotide sequences of the 12 mitochondrial protein-coding genes revealed that species with closer relationships share more identical gene blocks.

            Conclusion

            The two new mitochondrial genomes share many features, including gene contents, with other known nemertean mitochondrial genomes. The tRNA families display a composite substitution pathway. Gene order comparison to the proposed ground pattern of Bilateria and some lophotrochozoans suggests that the nemertean ancestral mitochondrial gene order most closely resembles the heteronemertean type. Phylogenetic analysis proposes a sister-group relationship between Hetero- and Hoplonemertea, which supports one of two recent alternative hypotheses of nemertean phylogeny.

            Keywords

            MtDNA Nemertea Nectonemertes mirabilis Zygeupolia rubens Phylogeny Gene rearrangement

            Background

            Knowledge of mitochondrial genomes is important for many scientific disciplines [1, 2] and the relative arrangement of mitochondrial genes has been effective for studying phylogenetic relationships [3, 4]. However, current knowledge of mtDNAs is uneven, and sequences available in GenBank are predominantly from vertebrate taxa. There are about 1275 described species [5] of nemerteans (ribbon worms, phylum Nemertea); these are mainly marine but terrestrial and freshwater species also are known. To date, complete mitochondrial genomes have been published for only three species in the phylum, Cephalothrix hongkongiensis (Palaeonemertea) [reported as Cephalothrix simula in [6]], Lineus viridis (Heteronemertea) [7], and Paranemertes cf. peregrina (Hoplonemertea)[8]. Nearly complete sequences exist for the palaeonemerteans Cephalothrix sp. [8] and Cephalothrix rufifrons [9]. Thus, current genomic knowledge of nemerteans is scant and taxon diversity is poorly sampled. In this study, we sequenced the complete mitochondrial genomes of two nemertean species, Nectonemertes cf. mirabilis (Hoplonemertea: Polystilifera) and Zygeupolia rubens (Heteronemertea). Mitochondrial gene arrangements, structures, and compositions, as well as translation and initiation codons and codon usage patterns, were compared with complete mtDNA sequences of other nemerteans. In addition, we compare gene order among Lophotrochozoa and we use the nucleotide sequences to analyze phylogenetic relationship among the included nemerteans.

            Results and discussion

            Genome organization and structure

            Genome composition and gene arrangement of Nectonemertes cf. mirabilis and Zygeupolia rubens are summarized in Figure 1 and Table 1. The mitochondrial genomes of N. cf. mirabilis and Z. rubens are circular DNA molecules of 15365 bp and 15513 bp, respectively. Lengths of the two nemertean mitochondrial genomes are within the range of previously sequenced nemertean mtDNAs - 14558 bp in Paranemertes cf. peregrina to 16296 bp in Cephalothrix hongkongiensis [6]. Both of the newly sequenced mitochondrial genomes contain 37 genes, including 13 protein-coding genes, two ribosomal RNAs, and 22 transfer RNAs. All genes except trnP and trnT are encoded on the same strand (Figure 1).
            http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-139/MediaObjects/12864_2011_4008_Fig1_HTML.jpg
            Figure 1

            Circular representation of the mtDNA of Nectonemertes cf. mirabilis and Zygeupolia rubens. Genes on the outer (H) strand are transcribed clockwise; those on the inner (L) strand are transcribed counter-clockwise. Transfer RNA genes are designated by the one-letter amino acid code for the corresponding amino acids; trnL1, trnL2, trnS1, and trnS2 differentiated on the basis of their codons CUN, UUR, UCN, and AGN, respectively. AT-rich non-coding region is represented in grey. The other small non-coding regions are not marked.

            Table 1

            Location of genes in the mitochondrial genomes of Nectonemertes cf. mirabilis and Zygeupolia rubens

            Nectonemertes cf. mirabilis

            Zygeupolia rubens

            Genes

            From 5'to 3'

            Size (bp)

            3'spacer a

            Genes

            From 5'to 3'

            Size (bp)

            3'spacer a

            trnY

            1-62

            62

            0

            trnY

            1-64

            64

            0

            trnP b

            125-63

            63

            2

            trnP b

            131-65

            67

            3

            nad6

            128-583

            456

            21

            nad6

            135-599

            465

            -8

            cob

            605-1741

            1137

            9

            cob

            592-1728

            1137

            -1

            trnS1 (UCN)

            1751-1811

            61

            -1

            trnS1(UCN)

            1728-1798

            71

            -1

            trnT b

            1876-1811

            66

            2

            trnT b

            1861-1798

            64

            2

            nad4L

            1879-2181

            303

            -7

            nad4L

            1864-2169

            306

            -7

            nad4

            2175-3536

            1362

            6

            nad4

            2163-3509

            1347

            1

            trnH

            3543-3602

            60

            0

            trnH

            3511-3574

            64

            2

            nad5

            3603-5348

            1746

            -1

            nad5

            3577-5308

            1732

            0

            trnE

            5348-5410

            63

            1

            trnE

            5309-5372

            64

            1

            trnG

            5412-5474

            63

            2

            trnG

            5374-5438

            65

            2

            cox3

            5477-6256

            780

            9

            cox3

            5441-6220

            780

            6

            trnK

            6266-6332

            67

            -2

            trnK

            6227-6287

            61

            -1

            trnA

            6331-6393

            63

            5

            trnA

            6287-6350

            64

            0

            trnF

            6399-6464

            66

            1

            trnF

            6351-6415

            65

            0

            trnQ

            6466-6532

            67

            0

            trnQ

            6416-6484

            69

            0

            trnR

            6533-6598

            66

            1

            trnR

            6485-6550

            66

            1

            trnN

            6600-6662

            63

            2

            trnN

            6552-6616

            65

            0

            trnI

            6665-6730

            66

            1

            trnI

            6617-6681

            65

            1

            nad3

            6732-7085

            354

            5

            nad3

            6683-7039

            357

            0

            cox1

            7091-8626

            1536

            12

            AT-rich

            7040-7877

            838

            0

            trnW

            8639-8703

            65

            0

            trnS2(AGN)

            7878-7949

            72

            0

            AT-rich

            8704-9405

            702

            0

            nad2

            7950-8957

            1008

            3

            trnS2 (AGN)

            9406-9473

            68

            -1

            cox1

            8961-10493

            1533

            0

            nad2

            9473-10480

            1008

            5

            trnW

            10494-10558

            65

            3

            cox2

            10486-11166

            681

            14

            cox2

            10562-11246

            685

            0

            trnD

            11181-11245

            65

            0

            trnD

            11247-11312

            66

            0

            atp8

            11246-11402

            157

            40

            atp8

            11313-11471

            159

            5

            atp6

            11443-12132

            700

            5

            atp6

            11477-12169

            693

            1

            trnC

            12138-12198

            61

            0

            trnC

            12171-12232

            62

            0

            trnM

            12199-12263

            65

            0

            trnM

            12233-12296

            64

            0

            rrnS

            12264-13068

            805

            0

            rrnS

            12297-13132

            836

            0

            trnV

            13069-13130

            62

            0

            trnV

            13133-13200

            68

            0

            rrnL

            13131-14308

            1178

            0

            rrnL

            13201-14448

            1248

            0

            trnL1(CUN)

            14309-14372

            64

            1

            trnL1(CUN)

            14449-14515

            67

            0

            trnL2(UUR)

            14374-14435

            62

            2

            trnL2(UUR)

            14516-14582

            67

            0

            nad1

            14438-15361

            924

            4

            nad1

            14583-15513

            931

            0

            aNegative numbers indicate that genes were overlapping

            bGenes coding in L strand

            For both species, protein-coding genes nad4L and nad4 share an overlap, by seven nucleotides, and nad6 overlaps cob by eight nucleotides in Z. rubens (Figure 1, Table 1). Such overlaps are common to all known mtDNA genomes of nemerteans [6, 8], and are found in many metazoan mtDNAs [10].

            Protein-coding genes

            Thirteen protein-coding genes (cox1-cox3, nad1-nad6, nad4L, cob, atp6, and atp8) were identified. Mitochondrial genomes often use a variety of nonstandard initiation codons [11]. Except for nad4 (GTG), nad5 (GTG), atp8 (GTG) and atp6 (GTT) in N. cf. mirabilis, and nad1 (GTG) and nad2 (GTG) in Z. rubens, the protein-coding genes of both species begin with ATG. The majority of genes in both species contain the full termination codon TAA or TAG, but some end with T (atp8 in N. cf. mirabilis, and nad5, cox2 and nad1 in Z. rubens). Such abbreviated stop codons are common among animal mitochondrial genes. In Z. rubens, the incomplete stop codons are immediately followed by the downstream tRNA gene (Figure 1, Table 1), whose secondary structure has been suggested to act as a signal for the cleavage of the polycistronic primary transcript [12, 13]. However, there also are direct junctions pairing ten protein-coding genes in N. cf. mirabilis (nad6/cob, nad4L/nad4, nad3/cox1, nad2/cox2, and atp8/atp6) and eight in Z. rubens (nad6/cob, nad4L/nad4, nad2/cox1 and atp8/atp6) (Figure 1, Table 1). Here, cleavage signals other than secondary structure of a tRNA gene may initiate processing of the polycistronic primary transcript [14]. For two protein-coding genes (nad6 and nad2) in both nemertean species and nad3 in N. cf. mirabilis, stem-loop structures were inferred to be at the 3' end and abutting the 5' end of the neighboring protein-coding gene, and may signal cleavage of the immature mRNA [15, 16].

            Transfer RNA and ribosomal RNA genes

            Both of the mitochondrial genomes encoded 22 tRNA genes found in other nemertean mtDNAs, which is typical of animal mitochondrial genomes [10]. They varied from 60 (trnH) to 68 (trnS2) nucleotides in N. cf. mirabilis and 61 (trnK) to 72 (trnS2) nucleotides in Z. rubens (Table 2); most were folded into a typical cloverleaf secondary structure (Figures 2, 3). The postulated tRNA cloverleaf structures generally contained 7 bp in the aminoacyl stem, 2 to 5 bp in the TψC stem, 5 bp in the anticodon stem, and 0 to 4 bp in the dihydrouridine (DHU) stem. Some tRNAs showed DHU-loop replacement (e.g., trnS1 of N. cf. mirabilis), as also found in L. viridis and P. cf. peregrina. In general, the lack of a DHU arm in two serine tRNAs is a common condition in metazoan mtDNAs [17]. The presence of such aberrant tRNA genes in mitochondrial genomes could be due to modification of tRNA secondary structure by replication slippage [18], or selection for mitochondrial genome minimization [19].
            Table 2

            Base composition of the mtDNA in six nemerteans

            Species

            Total nt

            T

            C

            A

            G

            A + T

            AT skew

            GC skew

            References

            Cephalothrix hongkongiensis

            16296

            47.4

            10.2

            27.5

            14.9

            74.9

            -0.266

            0.187

            [6]

            Cephalothrix sp.

            15800

            47.9

            10.0

            27.8

            14.3

            75.7

            -0.266

            0.178

            [8]

            Paranemertes cf. peregrina

            14558

            47.5

            10.0

            22.8

            19.7

            70.3

            -0.351

            0.322

            [8]

            Nectonemertes cf. mirabilis

            15365

            48.5

            10.5

            21.8

            19.2

            70.3

            -0.380

            0.293

            Present study

            Lineus viridis

            15388

            44.4

            11.9

            21.3

            22.4

            65.7

            -0.352

            0.306

            [7]

            Zygeupolia rubens

            15513

            45.0

            9.8

            21.0

            24.2

            66.0

            -0.364

            0.424

            Present study

            http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-139/MediaObjects/12864_2011_4008_Fig2_HTML.jpg
            Figure 2

            Secondary structure of tRNA families ( trnA - trnL1 ) in nemertean mtDNAs. The nucleotide substitution pattern for each tRNA family was modeled using as reference the structure determined for Nectonemertes cf. mirabilis.

            http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-139/MediaObjects/12864_2011_4008_Fig3_HTML.jpg
            Figure 3

            Secondary structure of tRNA families ( trnL2 - trnV ) in nemertean mtDNAs. The nucleotide substitution pattern for each tRNA family was modeled using as reference the structure determined for Nectonemertes cf. mirabilis.

            The mtDNAs of nemerteans investigated to date all have 20 tRNAs on the L strand and 2 tRNAs on the H strand ([69]). Secondary structures of nemertean tRNAs are presented and compared in Figures 2 and 3 (pattern follows [20]). Table 3 presents the tRNA lengths and the percent of identical nucleotides (%INUC) for the six nemerteans.
            Table 3

            Summary of multiple alignments of tRNA genes in nemertean mtDNAs

            ALN

            amino acid

            alignment

            length

            identical

            positions

            %INUC

            trnA

            Alanine

            72

            21

            29.17

            trnC

            Cysteine

            66

            39

            59.09

            trnD

            Aspartate

            66

            26

            39.39

            trnE

            Glutamate

            65

            27

            41.54

            trnF

            Phenylalanine

            68

            22

            32.35

            trnG

            Glycine

            67

            39

            58.21

            trnH

            Histidine

            67

            25

            37.31

            trnI

            Isoleucine

            72

            28

            38.89

            trnK

            Lysine

            73

            23

            31.51

            trnL1

            Leucine (CUN)

            69

            21

            30.43

            trnL2

            Leucine (UUR)

            68

            28

            41.18

            trnM

            Methionine

            66

            38

            57.58

            trnN

            Asparagine

            69

            20

            28.99

            trnP a

            Proline

            67

            24

            35.82

            trnQ

            Glutamine

            70

            28

            40.00

            trnR

            Arginine

            67

            16

            23.88

            trnS1

            Serine (UCN)

            71

            23

            32.39

            trnS2

            Serine (AGN)

            73

            30

            41.10

            trnT a

            Threonine

            71

            23

            32.39

            trnV

            Valine

            69

            33

            47.83

            trnW

            Tryptophan

            70

            27

            38.57

            trnY

            Tyrosine

            68

            32

            47.06

            ALN, alignment name; %INUC, percent of identical nucleotides

            agenes on the L strand

            Nucleotide conservation was strongest on the H strand, with trnC, trnG and trnM, having the highest levels of nucleotide conservation (%INUC > 50), followed by TrnE, trnL2, trnQ, trnS2, trnV and trnY at 40 ≤ %INUC ≤ 50 (Figure 2). The ten remaining tRNAs had %INUC values between 30 and 40; eight - trnD, trnF, trnH, trnI, trnK, trnL1, trnS1 and trnW - are located on the H strand, while two - trnP and trnT - are on the L strand. H-strand genes trnA, trnN and trnR had %INUC values ≤30.

            Conservation was positively H strand-biased, but no other pattern could be identified with respect to location of tRNAs along the genome. Two of the three most conserved tRNAs, trnC and trnM, are adjoining, while the third, trnG, adjoins the moderately conserved trnE and is relatively close to the three least conserved genes, trnA, trnN and trnR (Figure 1, Table 1). As observed by others (e.g., [20]), there was no self-evident link between abundance of codon families and the level of tRNA conservation, with the most abundant codon families (Leu2, Ile and Phe) not having the highest %INUCs (see below).

            A few mismatched nucleotide pairs (e.g., G-A, A-A, T-C, T-T) were found in the acceptor and/or the discriminator arms, without regard to the overall level of conservation of the tRNAs. As recently pointed out by Negrisolo et al. [20] for arthropods, metazoan mtDNAs commonly have such mismatches. It has been suggested that these may be corrected via RNA-editing mechanisms (e.g., [17]) or they may represent unusual pairings [21].

            Among the most conserved tRNAs in nemerteans, as in insects (e.g., [20]), nucleotide substitutions are mostly confined to TΨC and DHU loops and extra arms (Figures 2, 3), with 2-7 fully compensatory base changes (cbc; e.g., G-C vs. A-T) or hemi-cbcs (e.g., T-A vs. T-G) on acceptor and anticodon stems (see [20, 22]). As in insects [20], the number of cbcs and hemi-cbcs increased in stems as overall variation increased, especially in the TΨC stem.

            As found in insects, cbcs and hemi-cbcs characterized either single species or taxa at a higher taxonomic rank. An example of the first type is the A-T pair found in the trnC acceptor arm of P. cf. peregrina, which was mirrored by G-C in all other nemerteans (Figure 2). Few substitutions were present among C. hongkongiensis and Cephalothrix sp. (Figures 2, 3). An example of a full cbc characterizing a unique family is the A-T pair found in the acceptor stem of trnL1s of family Lineidae (L. viridis and Z. rubens), while other taxa exhibited the G-C pair (Figure 2). Similarly, a full cbc in the anticodon stem of trnG of two hoplonemerteans characterizes another high-taxonomic rank (Figure 2). Figures 2 and 3 depict several more examples. This points to the potential phylogenetic value of tRNA sequences, as demonstrated for other animal groups (e.g., [20, 23]), especially when secondary structures are taken into account [20]. While encouraging, clearly we need substantially more nemertean mitochondrial genomes to test this assertion for nemerteans.

            The anticodon usage of N. cf. mirabilis and Z. rubens was congruent with the corresponding tRNA genes of other nemerteans, with one exception. The anticodon of the trnS2 (AGN) gene in N. cf. mirabilis, P. cf. peregrina and three Cephalothrix species is GCT, but it is TCT in L. viridis and Z. rubens. Cameron et al. [24] found that anticodon changes in trnS2 (AGN) (GCT→TCT) must have occurred in the common ancestor of the insect clade Ischnocera, which was consistent with its phylogeny of lice. Similarly, this may constitute a kind of "rare genomic change" [25] in nemerteans and be a synapomorphy of Lineidae.

            As in all other metazoan mtDNAs sequenced to date, N. cf. mirabilis and Z. rubens mtDNAs contain genes for both small and large ribosomal subunit RNAs (rrnS and rrnL). Both genes are encoded by the same strand and are separated by trnV, as in many other metazoans. For N. cf. mirabilis and Z. rubens, respectively, the lengths of rrnL/rrnS are 1178/805 bp and 1248/836 bp, and the A + T contents are 75.5/72.4% and 70.9/70.5%.

            Base composition and codon usage

            The mtDNA of many invertebrates is characterized by a composition bias showing high values of A% and T% over G% and C%. The overall A + T content of N. cf. mirabilis and Z. rubens (70.3% and 66.0%, respectively) is consistent with those observed in other nemertean mitochondrial genomes. Though sample size for nemerteans is small, the A + T values appear to be linked in less (e.g., genus - e.g., Cephalothrix sp./C. hongkongiensis), as well as in more inclusive taxa (e.g., order - e.g., P. cf. peregrina/N. cf. mirabilis; L. viridis/Z. rubens) (Table 2). This might indicate a phylogenetic signal in nemerteans.

            Another feature of metazoan mtDNAs is asymmetry in nucleotide composition between the two strands, with one being rich in A and C, and the other being rich in T and G [26]. This asymmetry also is evident in the two nemertean mtDNA genomes here, with the genes encoded on the coding strand showing a strong bias toward T over A and toward G over C, as seen in the four other nemerteans, which have similar skewnesses (Table 2; Figure 4). This situation is common for mitochondrial genomes [26] and may be due to the presence of asymmetric patterns of mutational changes between strands [27, 28], and has been related with nucleotide deamination of DNA while transiently single-stranded during replication (this is not without controversy [29]) and/or transcription [30]. The relative importance of the two contributing processes (i.e., transcription vs. replication) remains to be assessed.
            http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-139/MediaObjects/12864_2011_4008_Fig4_HTML.jpg
            Figure 4

            Graphical representation of the percentage of A (black) and T (gray) across the whole mtDNA segment of six nemertean species (Accelrys). Y-axis values represent nucleotide %, calculated with a 100-bp sliding window using the program MacVector® 7.2.3; x-axis values represent the nucleotide positions corresponding to the linearized genome.

            We follow the pattern of [2] for displaying codon family abundance and relative synonymous codon usage (RSCU) for available nemertean protein-coding genes (Figures 5 and 6). To avoid bias due to incomplete stop codons, all stop codons are excluded from the analysis. The six nemertean mtDNAs use similar total numbers of non-stop codons (CDs), ranging from 3662 in P. cf. peregrina to 3707 in L. viridis. The codon families reveal a consistent pattern among the six nemertean species: the families with at least 50 CDs per thousand CDs (Leu1, Ile, Phe, Gly, Val) encompass an average 48.78% ± 1.33% of all CDs (Figure 5), with CDs rich in A + T favored over synonymous CDs of lower A + T content (Figure 6). For instance, the TTA codon accounts for a large majority of CDs in the Leu1 family. Whereas representation of the Leu1 (average = 77.3 ± 7.3%) and Leu2 (average = 22.7 ± 7.3%) codon families in nemertean protein-coding genes differs greatly, that of Ser1 (average = 60.8 ± 7.3%) and Ser2 (average = 39.2 ± 7.3%) is less extreme.
            http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-139/MediaObjects/12864_2011_4008_Fig5_HTML.jpg
            Figure 5

            Codon distribution in nemertean mtDNAs. CDspT, number of codons per thousands codons. Numbers to the right refer to the total number of codons.

            http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-139/MediaObjects/12864_2011_4008_Fig6_HTML.jpg
            Figure 6

            Relative Synonymous Codon Usage (RSCU) in nemertean mtDNAs. Codon families are provided on the x-axis, codons not present in the genome are orange colored.

            The invertebrate mitochondrial genome codes for 62 amino-acid codons [10]. As pointed out for Lepidoptera [2], the total number of codons used seems to be linked to the A + T content, which is the case among the six nemertean genomes analyzed. Thus, Cephalothrix sp. mtDNA has the highest (A + T)% content (see Table 2) and uses only 58 codons, never using the four codons rich in G + C (TCG, CGC, ACG, CGC) (Figure 6). Lineus viridis mtDNA uses all 62 codons and has the lowest A + T% among known nemertean genomes.

            The abundance of the four amino acid residues - Leu, Ile, Phe and Ser - is typical for invertebrate membrane proteins [2, 31], and they account here for more than 46.70% (average A + T = 50.14 ± 2.70%) of residues comprising the 13 mitochondrial proteins. The Leu and Ile amino acids share hydrophobic lateral chains.

            Two- and four-fold degenerate codon usage was similarly biased, with A/T favored over G/C in the third position (Figure 6) and in agreement with the AT-bias of protein-coding genes. Since the nemertean mitochondrial genome is AT-rich (Table 2), it can be expected that codons ending in A or T will predominate. From the overall RSCU values, it could be assumed that compositional constraints are the factor in shaping variation in codon usage among the genes in these mitochondrial genomes.

            Non-coding regions

            Metazoan mtDNAs usually have lengthy non-coding regions varying in size from ~100 bp to > 20 kbp [32, 33]. The mtDNAs of N. cf. mirabilis and Z. rubens contain a large number of unassigned nucleotides. There are 23 non-coding regions, with up to 855 nts, found throughout the N. cf. mirabilis mitochondrial genome. The AT-rich region located between the nad3 and trnS2 genes accounts for 838 nts and its AT content is 81.5%, which is higher than the remainder of the genome. Zygeupolia rubens has up to 879 non-coding nts distributed in 15 regions. The AT-rich region located between trnW and trnS2 genes is 702 nts and has an AT content of 74.9%, which also is higher than the remainder of the genome.

            In most metazoan mtDNAs, the largest non-coding region is thought to contain signals for replication and transcription, and is thus referred to as the control region [11]. The non-coding region has an increased AT composition, a characteristic typically used to identify origins of replication [10]. As in mtDNA genomes of other nemerteans, the AT-rich regions of N. cf. mirabilis and Z. rubens mtDNAs have the potential to form secondary structures such as stems and loops (Figure 7), which are thought to play an important role in the early stages of the replication and transcription process [34, 35]. Additionally, the AT-rich region in mtDNA of N. cf. mirabilis contains the tandemly repeated sequences (AAAAATATAAGATTTTTCAAATTCCAAAAATATAAAAT)3, (TTTTG)10, (TTTTTC)7, and several (A)n and (T)n homopolymer tracts. In mtDNAs of Z. rubens, we found the tandemly repeated sequences (GGGGGGGGGGGTAGTGTGGTTATGTTTTACTACACTCTTAGTAAAATATAAA)2, (TTTTTTG)10, and (TTTTTTTTA)6. Similar tandem repeat units within the largest non-coding regions also were found in the nemerteans Cephalothrix sp. [8], and C. hongkongiensis [6]. Tandem repeats are common within the control region of animal mtDNAs [34] and might be associated with regulatory mechanisms and recombination hot spots, and they might be the result of replication slippage events [36]. The high AT content and the predicted secondary structures of the AT-rich non-coding region of the N. cf. mirabilis and Z. rubens mtDNAs suggest that this region most likely contains the control region, though the control region in invertebrates, unlike that of vertebrates, is not well characterized and lacks discrete and conserved sequence blocks used in identification [37]. The nemertean mtDNA sequences examined here had multiple non-coding regions throughout their genomes. However, the location of the largest non-coding region is not conserved, and there is no obvious conservation of size (e.g., [6, 8]), nucleotide identities or potential secondary structures for the nemertean non-coding regions.
            http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-139/MediaObjects/12864_2011_4008_Fig7_HTML.jpg
            Figure 7

            Secondary structures predicted for the non-coding regions in the mitochondrial genome of two nemerteans. (A) Nectonemertes cf. mirabilis, AT-rich non-coding region between genes trnW and trnS2; (B, C) Zygeupolia rubens, AT-rich non-coding region between genes nad3 and trnS2.

            Gene order comparison

            Gene arrangements of the animal mitochondrial genome usually remain stable over long periods of evolutionary time, especially for protein-coding genes [10]. With some exceptions, mitochondrial gene order is relatively stable within major groups, and more variable between them [14]. This is the case for available nemertean mtDNA genomes, with mitochondrial genes transcribed from the same strand except for trnP and trnT. Among the three species of Cephalothrix (C. hongkongiensis, C. sp. and C. rufifrons), the gene order is identical for two but that of C. rufifrons differs from them. The two hoplonemertean species (P. cf. peregrina, N. cf. mirabilis) are identical to each other in gene order, as is the case for the two heteronemerteans (Z. rubens, L. viridis). The hoplo- and the heteronemertean species differ only by a translocation of the gene block S2/nad2 but they differ significantly from the three Cephalothrix species in the positions of atp8, nad6, nad2 and several tRNAs. The highest number of common intervals (1124) is between hoplo- and heteronemerteans, as indicated by results from CREx [38].

            We use two different gene sets, "all genes" and "non-tRNA genes" to compare the mt gene orders of nemerteans to the proposed ground pattern of Bilateria [39] and to mitochondrial gene orders of various lophotrochozoans: Terebratulina retusa (Brachiopoda) [40], Katharina tunicata (Mollusca)[14], Phoronis psammophila (Phoronida) [41], Perionyx excavatus (Annelida) [42], Urechis caupo (Annelida) [43] and Sipunculus nudus (Annelida)[44]. For the "all genes" set, all nemerteans share the adjacency nad4L/nad4 with the ground pattern of Bilateria and with the selected species (Figure 8). Nemerteans share the adjacencies rrnS/V/rrnL with Bilateria and the other species except U. caupo. The adjacency H/nad5 is shared by nemerteans and the selected species. Based on both gene sets, the hetero- and hoplonemerteans share the adjacency nad6/cob with K. tunicata [14], P. psammophila [41], P. excavatus [42], U. caupo [43], and S. nudus [44] and they share the adjacency atp8/atp6 with T. retusa, K. tunicata and the putative ground pattern of Bilateria (Figure 8; Additional file 1: Figure S1). These adjacencies may be a common plesiomorphic feature of Lophotrochozoa, such as Mollusca, Brachiopoda, and also Arthropoda mitochondrial genomes (e.g., [10]; [44]). However, neither of the latter two adjacencies is present in two Cephalothrix species, nor in the bryozoan Flustrellidra hispida [45].
            http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-139/MediaObjects/12864_2011_4008_Fig8_HTML.jpg
            Figure 8

            Mitochondrial gene order (all 37 genes) of Nemertea and selected lophotrochozoan species and the putative bilaterian ground pattern (according to [39]). Gene segments are not drawn to scale. All genes are transcribed from left to right except those in gray, which are transcribed from right to left. Unsequenced regions are in black. The adjacencies nad6/cob and atp8/atp6 are underlined. Previous gene orders from the following references: Cephalothrix [6, 8], Lineus [7], Paranemertes [8], Terebratulina [40], Katharina [14], Phoronis [41], Perionyx [42], Urechis [43], Sipunculus [44].

            In addition to visual comparison of genome maps, we analyzed gene order data with CREx [38], determining the number of common intervals. As shown in Table 4, the nemerteans share the highest number of common intervals (154, 184, 212) with K. tunicata and with P. psammophila (but this is a partial mitochondrial genome), while the lowest number was obtained in comparison with U. caupo (28, 18, 18). Although not significant, nemerteans and T. retusa, K. tunicata, and P. excavatus yield the highest numbers (18-20) in comparison with the putative bilaterian ground pattern.
            Table 4

            Pairwise common interval distance matrix of mitochondrial gene orders of nemerteans, the putative bilaterian ground pattern and six other lophotrochozoans *

            Common interval

            B

            P

            H

            H

            Tr

            Kt

            Uc

            Sn

            Pe

            Pp

            Bilaterian ground pattern (B)

            204\1326

            18

            20

            20

            18

            20

            12

            14

            20

            12

            Palaeonemertean (P)

            44

            204\1326

            108

            112

            40

            154

            28

            42

            38

            142

            Heteronemertean (H)

            52

            86

            204\1326

            1124

            68

            184

            18

            64

            56

            230

            Hoplonemertean (H)

            44

            72

            178

            204\1326

            84

            212

            18

            68

            66

            254

            Terebratulina retusa (Tr)

            52

            86

            204

            178

            204\1326

            128

            20

            74

            82

            110

            Katharina tunicata (Kt)

            48

            56

            106

            94

            106

            204\1326

            20

            62

            64

            266

            Urechis caupo (Uc)

            16

            8

            14

            8

            14

            34

            204\1326

            54

            144

            22

            Sipunculus nudus (Sn)

            34

            12

            22

            16

            22

            26

            26

            204\1326

            158

            38

            Perionyx excavatus (Pe)a

            28

            24

            40

            32

            40

            48

            44

            60

            204\1254

            44

            Phoronis psammophila (Pp)b

            40

            48

            84

            76

            84

            98

            22

            24

            38

            204\864

            *bold numbers represent pairwise common interval distances between mitochondrial gene orders (37 genes in total), while italic numbers represent pairwise common interval distances between mt gene orders without tRNAs (15 genes in total)

            alacks trnR

            blacks several tRNAs

            Figure 8 shows tRNA genes change relative position much faster than the protein-coding and rRNA genes, as reported from previous studies (e.g., [46, 47]).

            Excluding tRNAs, the gene order of heteronemerteans is identical to that of T. retusa [40] and some gastropods, e.g., Conus textile [48], Ilyanassa obsoleta [49], Thais clavigera [37] and Lophiotoma cerithiformis [50]. Other molluscs, like the polyplacophoran K. tunicata [14], the gastropod Haliotis rubra [51] and the cephalopod Octopus vulgaris [52] show a similar gene order, but are distinguished by a large inversion of about half the mt genome (Additional file 1: Figure S1). Without tRNAs, heteronemerteans and T. retusa, which has the same gene order, share the greatest number of possible common intervals (204) (Table 4), and both share the greatest number (52) with the putative bilaterian ground pattern.

            We also determined breakpoints and reversal distances between these taxa with the two gene sets (Additional files 2, 3: Tables S1, S2). For "all genes", hetero- and hoplonemerteans share the same breakpoint distance (31) and the same reversal distance (28) (whereas palaeonemerteans are 32 and 31, respectively) with respect to the putative bilaterian ground pattern. Heteronemerteans have the lowest values among the nemerteans when tRNAs are excluded from the analysis. These comparisons with the putative bilaterian ground pattern and with other lophotrochozoans gene orders (especially when excluding tRNAs), suggest that the heteronemertean gene order is likely to be closest to the ancestral mitochondrial gene order of Nemertea. This is in agreement with a previous study [7].

            Phylogenetic analysis

            We performed a phylogenetic analysis based on nucleotide sequences of protein-coding genes to better understand relationships within the Nemertea. The phylogenetic tree in Figure 9, reconstructed by maximum likelihood and Bayesian analyses, indicates that similar gene arrangements reflect close phylogenetic affinity. This supports previous hypotheses that Hoplonemertea and Heteronemertea are sister taxa (e.g., [5355]). However, a phylogenetic analysis based on amino acid sequences (data not shown) suggests Hoplonemertea as sister group to Palaeonemertea. This contradicts many but not all previous analyses (e.g., [55]). We consider it unsupported by our data, given the low Bayesian posterior probability (0.61) for this clade. This suggests, however, that amino acid sequence data deserve continued attention in future analyses with new, additional data.
            http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-139/MediaObjects/12864_2011_4008_Fig9_HTML.jpg
            Figure 9

            Best tree from the Maximum Likelihood analysis with 5921 nt (first and second codon positions) of protein-coding genes. Node support is indicated above (Bayesian posterior probabilities) and below (maximum likelihood bootstrap values) each branch. A Bayesian analysis resulted in the same species topology.

            Conclusion

            To date, complete or nearly complete mtDNA sequences have been determined for seven nemerteans, a very small sampling compared to those available for vertebrates or arthropods. The two new mtDNA genomes, for Nectonemertes cf. mirabilis and Zygeupolia rubens, share substantial similarity with those of other nemertean mitochondrial genomes, and gene content and A + T richness is similar to those common for animal mtDNAs.

            There is strong skew in the distribution of nucleotides between the two strands.

            The evolution of nemertean tRNAs seems to have been variable both in terms of sequence conservation and nucleotide substitution processes. The presence of full and hemi-cbcs characterizing taxa at different taxonomic levels may indicate the potential phylogenetic value of tRNA sequences.

            Nemertean mtDNAs are punctuated by non-coding portions highly variable in size. Among them, the AT-rich non-coding region, which appears to be a fast-evolving genomic region characterized by short to medium-size repeated motifs/AT-rich patterns, may be associated with the initiation of replication or transcription.

            Phylogenetic analysis supports the close phylogenetic affinities in nemerteans one might infer from similarities in gene arrangements, with Hetero- and Hoplonemerteans as sister-clades. Gene order analysis suggests that the heteronemertean pattern most closely resembles the likely ancestral condition among nemerteans, which is counterintuitive in light of the phylogenetic analysis. Confidence that we understand evolution of the nemertean mitochondrial genome is likely to require investigating many more nemertean mtDNAs, especially a full representation of palaeonemertean diversity.

            Methods

            DNA extraction, PCR and sequencing

            Specimens were collected off Point Conception, California (Nectonemertes cf. mirabilis) and at Fort Pierce, Florida (Zygeupolia rubens), USA. We use the "cf." qualifier to confer reasonable caution that the Pacific worm traditionally designated N. mirabilis (see [56]) is conspecific with its presumed cognate originally described from the North Atlantic Ocean. Samples were frozen in liquid nitrogen and preserved in RNAlater. Total DNA was extracted from a single individual specimen using the DNeasy Tissue Kit following the manufacturer's protocol (Qiagen, Valencia, CA, USA). PCR primers used for amplification are listed in Table 5. Preliminary nemertean-specific primers (N12SF, N16SR, NCOX2R) were designed based on sequence alignment of four mitochondrial genome sequences (Cephalothrix hongkongiensis, Cephalothrix. sp., Lineus viridis, and Paranemertes cf. peregrina) retrieved from Genbank. For both species, the partial regions rrnS-rrnL and rrnL-cob were amplified first. For N. cf. mirabilis, partial fragments of cox1 and cox3 genes were amplified using universal PCR primers LCO-2198/HCO-1490, cox3F/cox3R ([59]; [9]). These sequences were used to design specific primers to amplify the remaining mitochondrial genome fragments (cob-cox3, cox3-cox1 and cox1-rrnS). For Z. rubens, the fragment of cox1-cox2 was amplified using the universal primer LCO-2198 [59] combined with the specific primer NCOX2R. Based on sequences obtained, specific primers were designed to amplify the regions cox2-rrnS, cob-cox3 and cox3-cox1. Conventional PCR and long PCR, cloning, and sequencing were performed as described in Chen et al. [6, 8].
            Table 5

            PCR primers used to amplify the mitochondrial genomes of Nectonemertes cf. mirabilis

            Primer name

            Sequence (5' → 3')

            References

            Universal

              

            rrnS-rrnL

              

            N12SF

            TGTGCCAGCTTCCGCGGTTATAC

            Present study

            N16SR

            ACGCTGTTATCCCTATGGTA

            Present study

            rrnL-cob

              

            16SarL

            CGCCTGTTTATCAAAAACAT

            [57]

            CytbR

            GCRTAWGCRAAWARRARTAYCAYTCWGG

            [58]

            Nectonemertes cf. mirabilis

              

            cox1

              

            LCO-1490

            GGTCAACAAATCATAAAGATATTGG

            [59]

            HCO-2198

            TAAACTTCAGGGTGACCAAAAAATCA

            [59]

            cox3

              

            cox3F

            TGCGWTGAGGWATAATTTTATTTATT

            [8]

            cox3R

            ACCAAGCAGCTGCTTCAAAACCAAA

            [8]

            cob-cox3

              

            Nm cobF

            TCGGTGGATAATGCTACTTTG

            Present study

            Nm COX3R

            ACCAGAAGCCAACAATACAGC

            Present study

            cox3-cox1

              

            Nm COX3F

            TGTTGGCTTCTGGTGTTAGTG

            Present study

            Nm COX1R

            GAGCCTCTTTCAACAACAGCA

            Present study

            cox1-rrnS

              

            NmCOX1F

            AATCTGGTCTGGGTTGGTTGGCACTGCGTTA

            Present study

            Nm12SR

            GACTCCCCTGAAAGGACATAAAACACCG

            Present study

            Zygeupolia rubens

              

            cob-cox3

              

            Zrcob F

            CTTTGGGTTTGTTGCTGTTG

            Present study

            ZrCOX3R

            GTTGAACCATAAATCCCATC

            Present study

            cox3-cox1

              

            cox3F

            TGCGWTGAGGWATAATTTTATTTATT

            [8]

            ZrCOX1R

            GAGCCTCTTTCAACAACAGCA

            Present study

            cox1-cox2

              

            LCO-1490

            GGTCAACAAATCATAAAGATATTGG

            [59]

            NCOX2R

            AAAGAATGATTWGCWCCAC

            Present study

            cox2-rrnS

              

            ZrCOX2F

            TTTGGCTTTACCTTCTTTGC

            Present study

            Zr12SR

            AAATAAGACACCGCCAAGT

            Present study

            Sequence assemblage and annotation

            All sequences were checked and assembled by visual inspection using the program SeqMan (DNA star, Madison, WI, USA). Protein-coding genes and ribosomal RNA genes were identified by their similarity to previously reported mitochondrial genomes of Cephalothrix hongkongiensis (GenBank #NC_012821), C. rufifrons (EF140788), Cephalothrix sp. (NC_014869), Lineus viridis (NC_012889), and Paranemertes cf. peregrina (NC_014865). Most tRNAs were identified by using invertebrate mitochondrial codon predictors with tRNAscan-SE 1.21 [60]. The remaining tRNA genes were found by inspecting sequences for tRNA-like secondary structures and anticodons. Multiple alignments of tRNA genes were produced, and the percent of identical nucleotides (%INUC) was calculated for six nemertean tRNA sequences. Secondary structures within the non-coding fragments were visualized by using RnaViz 2.0 [61], and the mitochondrial genome was visualized using CGView [62].

            Genomics analysis

            Nucleotide composition and Relative Synonymous Codon Usage (RSCU) values were analyzed with MEGA 4.0 [63]. AT- and GC-skew were determined by using the formulation of [26].

            Gene order comparisons

            Gene orders were compared between all available nemerteans (see above), the putative bilaterian ground pattern [39], Terebratulina retusa [40], Katharina tunicata [14], Phoronis psammophila [41], Perionyx excavatus [42], Urechis caupo [43] and Sipunculus nudus [44].

            The gene orders were compared with two different gene sets: "all genes" included all 37 mitochondrial genes, whereas "non-tRNA genes" included only the two ribosomal genes and the 13 protein-coding genes.

            The differences between gene orders were analysed using common intervals [38], breakpoints [64] and reversal distances [65] implemented in the CREx tool [38].

            Phylogenetic analysis

            The currently available near-complete and complete mitochondrial nemertean genome data (Cephalothrix sp., C. hongkongiensis, L. viridis, and P. cf. peregrina, but not the partial genome sequence of C. rufifrons) were combined with sequences from this study for phylogenomic analyses. The nucleic acids for all 12 protein-coding genes (except atp8, which is shortest and least conserved between the taxa) were aligned using Clustal X [66] with the default settings. Ambiguously aligned portions of both alignments were excluded using Gblocks version 0.91b [67] with default block parameters. We also excluded third codon positions because of the fast substitution rate. The total number of nucleotides used for the phylogenetic analysis was 5921.

            Based on previous studies of metazoan relationships (e.g., [6873]), the following six species were selected as outgroups: a mollusc (Katharina tunicata), a brachiopod (Terebratalia retusa), a phoronid (Phoronis psammophila), and three annelids (Perionyx excavatus, Sipunculus nudus and Urechis caupo).

            Phylogenetic trees were estimated under maximum likelihood (ML) and Bayesian inference (BI). ML analysis on the combined nucleotide dataset alignments was performed in RAxML 7.2.7 [74, 75] available on the CIPRES web portal [76] with the GTRGAMMA substitution model. Support was estimated by performing 1000 bootstrap replicates. BI analysis was performed with MrBayes version 3.0b4 [77, 78], using GTR + I + G model, a best-fit model selected by MrModeltest v2.2 [79] following the Akaike information criterion (AIC). The Monte Carlo Markov chain (MCMC) length was 1,000,000 generations and sampled every 100 generations. The first 2500 trees from each run were discarded as a burn-in.

            Amino acid sequences were aligned with both Clustal X [66] and MAFFT using the G-INS-i strategy [80]. BI analyses were performed with MrBayes version 3.0b4 [77, 78] with the mtRev + I + G model, selected by Protest 10.2 [81] as optimal. We also implemented the CAT + GTR model in PhyloBayes 3 [82]. The ML analysis was carried out with RAxML 7.2.7 [74, 75] with CAT model.

            The mitochondrial genome sequences of N. cf. mirabilis and Z. rubens are deposited in GenBank under the accession numbers HQ997772 and HQ997773.

            Abbreviations

            atp6 and atp8

            ATP synthase subunits 6 and 8

            cob

            cytochrome b

            cox1-3

            subunits I-III of cytochrome c oxidase

            nad1-6 and nad4L

            NADH dehydrogenase subunits 1-6 and 4 L

            rrnL and rrnS

            the large and small subunits of ribosomal RNA

            trnX

            genes encoding for transfer RNA molecules with corresponding amino acids denoted by the one-letter code and codon indicated in parentheses (xxx) when necessary

            DHU: 

            dihydrouridine loop

            MtDNA: 

            mitochondrial DNA

            NC: 

            non-coding region

            PCR: 

            polymerase chain reaction

            Kb: 

            kilobase

            bp: 

            base pair

            nt: 

            nucleotide

            nucleotide symbol combination: 

            R = A/G; Y = C/T; W = A/T; K = G/T; N = A/G/C/T

            Declarations

            Acknowledgements

            This work was supported by the National Natural Science Foundation of China (to SCS, grant no. 30970333,), the Swedish Research Council (to PS), Smithsonian Institution Scholarly Studies, Research Opportunities, and Marine Science Network awards (to JLN) and represents contribution 878 of the Smithsonian Marine Station at Fort Pierce. JLN is grateful to James Childress (UCSB) and his support from the US National Science Foundation for the opportunity to collect living Nectonemertes cf. mirabilis.

            Authors’ Affiliations

            (1)
            Department of Biological and Environmental Sciences, University of Gothenburg
            (2)
            Institute of Evolution & Marine Biodiversity, Ocean University of China
            (3)
            Department of Rheumatology and Inflammation Research, Sahlgrenska Academy, University of Gothenburg
            (4)
            Department of Invertebrate Zoology, National Museum of Natural History, Smithsonian Institution

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