Scholarly article on topic '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'

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 Academic research paper on "Biological sciences"

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Academic research paper on topic "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"


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

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

* Correspondence:

4Department of Invertebrate Zoology, NationalMuseum of NaturalHistory, Smithsonian Institution, Washington, DC 20560-0163, USA Fulllist of author information is available at the end of the article

O© 2012 Chen et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons "WlVhMl Central Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Hai-Xia Chen1,2, Shi-Chun Sun2, Per Sundberg1, Wei-Cheng Ren3 and Jon L Norenburg'



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: Polystili-fera) and Zygeupolia rubens (Heteronemertea). Mito-chondrial 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 Lophotrocho-zoa 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 Nectone-mertes 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).

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].

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; trnL 1, 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.

Protein-coding genes

Thirteen protein-coding genes (cox1-cox3, nad1-nad6, nad4L, cob, atp6, and atp8) were identified. Mitochon-drial 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

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'spacera Genes From 5'to 3' Size (bp) 3'spacera

trnY 1-62 62 0 trnY 1-64 64 0

trnPb 125-63 63 2 trnPb 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

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 clover-leaf secondary structure (Figures 2, 3). The postulated

tRNA cloverleaf structures generally contained 7 bp in the aminoacyl stem, 2 to 5 bp in the TyC 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., trnSl of N. cf. mirabilis), as also found in L.

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

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].

The mtDNAs of nemerteans investigated to date all have 20 tRNAs on the L strand and 2 tRNAs on the H strand ([6-9]). 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.

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, trnLl, trnSl 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 TYC 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 TYC 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 trnLls 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 phy-logenetic 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 nemer-tean 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 phylo-geny of lice. Similarly, this may constitute a kind of "rare genomic change" [25] in nemerteans and be a synapomorphy of Lineidae.

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.

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%.

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.

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 nemer-teans is small, the A + T values appear to be linked in

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

trnPa 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

trnTa 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

less (e.g., genus - e.g., Cephalothrix sp./C. hongkongien-sis), 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 skew-nesses (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.

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

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.

of non-stop codons (CDs), ranging from 3662 in P. cf. (Leul, Ile, Phe, Gly, Val) encompass an average 48.78%

peregrina to 3707 in L. viridis. The codon families reveal ± 1.33% of all CDs (Figure 5), with CDs rich in A + T

a consistent pattern among the six nemertean species: favored over synonymous CDs of lower A + T content

the families with at least 50 CDs per thousand CDs (Figure 6). For instance, the TTA codon accounts for a

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.

large majority of CDs in the Leul family. Whereas representation of the Leul (average = 77.3 ± 7.3%) and Leu2 (average = 22.7 ± 7.3%) codon families in nemertean protein-coding genes differs greatly, that of Serl

(average = 60.8 ± 7.3%) and Ser2 (average = 39.2 ± 7.3%) is less extreme.

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 mitochon-drial 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

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.

(AAAAATATAAGATTTTTCAAATTCCAAAAATA-TAAAAT)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 (GGGGGGGGGGGTAGTGTGGTTATGTTTTACTA-CACTCTTAGTAAAATATAAA)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.

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. vir-idis). 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 nemer-teans to the proposed ground pattern of Bilateria [39] and to mitochondrial gene orders of various lophotro-chozoans: Terebratulina retusa (Brachiopoda) [40], Katharina tunicata (Mollusca)[14], Phoronis psammo-phila (Phoronida) [41], Perionyx excavatus (Annelida) [42], Urechis caupo (Annelida) [43] and Sipunculus nudus (Annelida)[44]. For the "all genes" set, all nemer-teans 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 Mol-lusca, 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].

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 mitochon-drial 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.

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 clavi-gera [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, heterone-merteans 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. Het-eronemerteans 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 het-eronemertean 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 Heterone-mertea are sister taxa (e.g., [53-55]). However, a phyloge-netic analysis based on amino acid sequences (data not shown) suggests Hoplonemertea as sister group to Palaeo-nemertea. 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.


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

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].

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

arthropods. The two new mtDNA genomes, for Nectone-mertes 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 taxo-nomic 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 Hoplonemer-teans 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 nemer-tean mitochondrial genome is likely to require investigating many more nemertean mtDNAs, especially a full representation of palaeonemertean diversity.


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 conspe-cific 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 hon-gkongiensis, Cephalothrix. sp., Lineus viridis, and Para-nemertes cf. peregrina) retrieved from Genbank. For both species, the partial regions rrnS-rrnL and rrnL-cob

Cephalothrix sp. Cephalothrix hongkongiensis

-1 i ne us viridis



■ Zygeupolia rubens

-Nectonemertes cf. mirabilis

-Paranemertes cf. peregrina

Katharina tunica ta

- Phoronis psammophila

-Sipunculus nudus

i-Perionyx excavatus



Urechis caupo - Terebratulina retusa

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.

Table 5 PCR primers used to amplify the mitochondrial genomes of Nectonemertes cf. mirabilis

Primer name Sequence (5' ® В') References








Nectonemertes cf. mirabilis














Zygeupolia rubens













were amplified first. For N. cf. mirabilis, partial fragments of coxl and cox3 genes were amplified using universal PCR primers LC0-2198/HC0-1490, cox3F/cox3R ([59]; [9]). These sequences were used to design specific primers to amplify the remaining mitochondrial genome fragments (cob-cox3, cox3-cox1 and coxl-rrnS). For Z. rubens, the fragment of cox1-cox2 was amplified using the universal primer LCO-2198 [59] combined with the specific primer NC0X2R. 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].

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 nemer-tean 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 phyloge-nomic 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., [68-73]), 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.

Additional material

Additional file 1: Figure S1. Mitochondrialgene order (protein-coding genes and rRNAs only) of Nemertea and selected lophotrochozoan species and the putative bilaterian ground pattern (according to [39]). Gene segments are not drawn to scale. Allgenes are transcribed from left-to-right except those in gray, which are transcribed from right to left. The adjacencies nad6/cob and atp8/atp6 are underlined. The translocation of nad2 in the heteronemerteans and hoplonemerteans is highlight by *. Gene orders according to the following references: Cephalothrix [6,8], Lineus [7], Paranemertes [8], Terebratulina [40], Katharina [14], Phoronis [41], Perionyx [42], Urechis [43], Sipunculus [44].

Additional file 2: Table S1. Pairwise breakpoint distance matrix of mitochondrial gene orders of nemerteans, the bilaterian ground pattern and six other lophotrochozoans*

Additional file 3: Table S2. Pairwise reversaldistance matrix of mitochondrialgene orders of nemerteans, the bilaterian ground pattern and six other lophotrochozoans*


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 ribosomalRNA; 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: mitochondrialDNA; 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.


This work was supported by the NationalNaturalScience 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 gratefulto James Childress (UCSB) and his support from the US NationalScience Foundation for the opportunity to collect living Nectonemertes cf. mirabilis.

Author details

department of Biologicaland EnvironmentalSciences, University of Gothenburg, PO Box 463, SE-405 30 Gothenburg, Sweden. 2Institute of Evolution & Marine Biodiversity, Ocean University of China, 5 Yushan Road, Qingdao 266003, China. 3Department of Rheumatology and Inflammation Research, Sahlgrenska Academy, University of Gothenburg, PO Box 480, SE-405 30, Sweden. 4Department of Invertebrate Zoology, NationalMuseum of NaturalHistory, Smithsonian Institution, Washington, DC 20560-0163, USA.

Authors' contributions

HXC performed the majority of the molecular experiments and analyzed the data, and drafted the manuscript. SCS supervised the research. PS contributed to the analysis of the data. WCR performed part of the study, and provided technical assistance. JLN collected specimens, conceived, designed the research plan and did significant revisions of the manuscript draft. Allauthors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Received: 2 May 2011 Accepted: 17 April 2012 Published: 17 April 2012


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Cite this article as: Chen et al.: 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. BMC Genomics 2012


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