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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">A comparative study of nemertean complete mitochondrial genomes, including two new ones for <italic>Nectonemertes </italic>
cf. <italic>mirabilis </italic>
and <italic>Zygeupolia rubens</italic>
, may elucidate the fundamental pattern for the phylum Nemertea</title>
<author><name sortKey="Chen, Hai Xia" sort="Chen, Hai Xia" uniqKey="Chen H" first="Hai-Xia" last="Chen">Hai-Xia Chen</name>
<affiliation><nlm:aff id="I1">Department of Biological and Environmental Sciences, University of Gothenburg, PO Box 463, SE-405 30 Gothenburg, Sweden</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="I2">Institute of Evolution & Marine Biodiversity, Ocean University of China, 5 Yushan Road, Qingdao 266003, China</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Sun, Shi Chun" sort="Sun, Shi Chun" uniqKey="Sun S" first="Shi-Chun" last="Sun">Shi-Chun Sun</name>
<affiliation><nlm:aff id="I2">Institute of Evolution & Marine Biodiversity, Ocean University of China, 5 Yushan Road, Qingdao 266003, China</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Sundberg, Per" sort="Sundberg, Per" uniqKey="Sundberg P" first="Per" last="Sundberg">Per Sundberg</name>
<affiliation><nlm:aff id="I1">Department of Biological and Environmental Sciences, University of Gothenburg, PO Box 463, SE-405 30 Gothenburg, Sweden</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Ren, Wei Cheng" sort="Ren, Wei Cheng" uniqKey="Ren W" first="Wei-Cheng" last="Ren">Wei-Cheng Ren</name>
<affiliation><nlm:aff id="I3">Department of Rheumatology and Inflammation Research, Sahlgrenska Academy, University of Gothenburg, PO Box 480, SE-405 30, Sweden</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Norenburg, Jon L" sort="Norenburg, Jon L" uniqKey="Norenburg J" first="Jon L" last="Norenburg">Jon L. Norenburg</name>
<affiliation><nlm:aff id="I4">Department of Invertebrate Zoology, National Museum of Natural History, Smithsonian Institution, Washington, DC 20560-0163, USA</nlm:aff>
</affiliation>
</author>
</titleStmt>
<publicationStmt><idno type="wicri:source">PMC</idno>
<idno type="pmid">22507536</idno>
<idno type="pmc">3368773</idno>
<idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3368773</idno>
<idno type="RBID">PMC:3368773</idno>
<idno type="doi">10.1186/1471-2164-13-139</idno>
<date when="2012">2012</date>
<idno type="wicri:Area/Pmc/Corpus">000492</idno>
</publicationStmt>
<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">A comparative study of nemertean complete mitochondrial genomes, including two new ones for <italic>Nectonemertes </italic>
cf. <italic>mirabilis </italic>
and <italic>Zygeupolia rubens</italic>
, may elucidate the fundamental pattern for the phylum Nemertea</title>
<author><name sortKey="Chen, Hai Xia" sort="Chen, Hai Xia" uniqKey="Chen H" first="Hai-Xia" last="Chen">Hai-Xia Chen</name>
<affiliation><nlm:aff id="I1">Department of Biological and Environmental Sciences, University of Gothenburg, PO Box 463, SE-405 30 Gothenburg, Sweden</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="I2">Institute of Evolution & Marine Biodiversity, Ocean University of China, 5 Yushan Road, Qingdao 266003, China</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Sun, Shi Chun" sort="Sun, Shi Chun" uniqKey="Sun S" first="Shi-Chun" last="Sun">Shi-Chun Sun</name>
<affiliation><nlm:aff id="I2">Institute of Evolution & Marine Biodiversity, Ocean University of China, 5 Yushan Road, Qingdao 266003, China</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Sundberg, Per" sort="Sundberg, Per" uniqKey="Sundberg P" first="Per" last="Sundberg">Per Sundberg</name>
<affiliation><nlm:aff id="I1">Department of Biological and Environmental Sciences, University of Gothenburg, PO Box 463, SE-405 30 Gothenburg, Sweden</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Ren, Wei Cheng" sort="Ren, Wei Cheng" uniqKey="Ren W" first="Wei-Cheng" last="Ren">Wei-Cheng Ren</name>
<affiliation><nlm:aff id="I3">Department of Rheumatology and Inflammation Research, Sahlgrenska Academy, University of Gothenburg, PO Box 480, SE-405 30, Sweden</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Norenburg, Jon L" sort="Norenburg, Jon L" uniqKey="Norenburg J" first="Jon L" last="Norenburg">Jon L. Norenburg</name>
<affiliation><nlm:aff id="I4">Department of Invertebrate Zoology, National Museum of Natural History, Smithsonian Institution, Washington, DC 20560-0163, USA</nlm:aff>
</affiliation>
</author>
</analytic>
<series><title level="j">BMC Genomics</title>
<idno type="eISSN">1471-2164</idno>
<imprint><date when="2012">2012</date>
</imprint>
</series>
</biblStruct>
</sourceDesc>
</fileDesc>
<profileDesc><textClass></textClass>
</profileDesc>
</teiHeader>
<front><div type="abstract" xml:lang="en"><sec><title>Background</title>
<p>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: <italic>Nectonemertes </italic>
cf. <italic>mirabilis </italic>
and <italic>Zygeupolia rubens</italic>
.</p>
</sec>
<sec><title>Results</title>
<p>The sizes of the entire mitochondrial genomes are 15365 bp for <italic>N</italic>
. cf. <italic>mirabilis </italic>
and 15513 bp for <italic>Z. rubens</italic>
. 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 <italic>N</italic>
. cf. <italic>mirabilis </italic>
is identical to that of <italic>Paranemertes </italic>
cf. <italic>peregrina</italic>
, both of which are Hoplonemertea, while that of <italic>Z. rubens </italic>
is the same as in <italic>Lineus viridis</italic>
, 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.</p>
</sec>
<sec><title>Conclusion</title>
<p>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.</p>
</sec>
</div>
</front>
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<pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">BMC Genomics</journal-id>
<journal-id journal-id-type="iso-abbrev">BMC Genomics</journal-id>
<journal-title-group><journal-title>BMC Genomics</journal-title>
</journal-title-group>
<issn pub-type="epub">1471-2164</issn>
<publisher><publisher-name>BioMed Central</publisher-name>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">22507536</article-id>
<article-id pub-id-type="pmc">3368773</article-id>
<article-id pub-id-type="publisher-id">1471-2164-13-139</article-id>
<article-id pub-id-type="doi">10.1186/1471-2164-13-139</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject>
</subj-group>
</article-categories>
<title-group><article-title>A comparative study of nemertean complete mitochondrial genomes, including two new ones for <italic>Nectonemertes </italic>
cf. <italic>mirabilis </italic>
and <italic>Zygeupolia rubens</italic>
, may elucidate the fundamental pattern for the phylum Nemertea</article-title>
</title-group>
<contrib-group><contrib contrib-type="author" id="A1"><name><surname>Chen</surname>
<given-names>Hai-Xia</given-names>
</name>
<xref ref-type="aff" rid="I1">1</xref>
<xref ref-type="aff" rid="I2">2</xref>
<email>chen_hai_xia@126.com</email>
</contrib>
<contrib contrib-type="author" id="A2"><name><surname>Sun</surname>
<given-names>Shi-Chun</given-names>
</name>
<xref ref-type="aff" rid="I2">2</xref>
<email>sunsc@ouc.edu.cn</email>
</contrib>
<contrib contrib-type="author" id="A3"><name><surname>Sundberg</surname>
<given-names>Per</given-names>
</name>
<xref ref-type="aff" rid="I1">1</xref>
<email>per.sundberg@zool.gu.se</email>
</contrib>
<contrib contrib-type="author" id="A4"><name><surname>Ren</surname>
<given-names>Wei-Cheng</given-names>
</name>
<xref ref-type="aff" rid="I3">3</xref>
<email>renweicheng1981@126.com</email>
</contrib>
<contrib contrib-type="author" corresp="yes" id="A5"><name><surname>Norenburg</surname>
<given-names>Jon L</given-names>
</name>
<xref ref-type="aff" rid="I4">4</xref>
<email>norenburgj@si.edu</email>
</contrib>
</contrib-group>
<aff id="I1"><label>1</label>
Department of Biological and Environmental Sciences, University of Gothenburg, PO Box 463, SE-405 30 Gothenburg, Sweden</aff>
<aff id="I2"><label>2</label>
Institute of Evolution & Marine Biodiversity, Ocean University of China, 5 Yushan Road, Qingdao 266003, China</aff>
<aff id="I3"><label>3</label>
Department of Rheumatology and Inflammation Research, Sahlgrenska Academy, University of Gothenburg, PO Box 480, SE-405 30, Sweden</aff>
<aff id="I4"><label>4</label>
Department of Invertebrate Zoology, National Museum of Natural History, Smithsonian Institution, Washington, DC 20560-0163, USA</aff>
<pub-date pub-type="collection"><year>2012</year>
</pub-date>
<pub-date pub-type="epub"><day>17</day>
<month>4</month>
<year>2012</year>
</pub-date>
<volume>13</volume>
<fpage>139</fpage>
<lpage>139</lpage>
<history><date date-type="received"><day>2</day>
<month>5</month>
<year>2011</year>
</date>
<date date-type="accepted"><day>17</day>
<month>4</month>
<year>2012</year>
</date>
</history>
<permissions><copyright-statement>Copyright ©2012 Chen et al; licensee BioMed Central Ltd.</copyright-statement>
<copyright-year>2012</copyright-year>
<copyright-holder>Chen et al; licensee BioMed Central Ltd.</copyright-holder>
<license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/2.0"><license-p>This is an Open Access article distributed under the terms of the Creative Commons Attribution License (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/2.0">http://creativecommons.org/licenses/by/2.0</ext-link>
), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p>
</license>
</permissions>
<self-uri xlink:href="http://www.biomedcentral.com/1471-2164/13/139"></self-uri>
<abstract><sec><title>Background</title>
<p>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: <italic>Nectonemertes </italic>
cf. <italic>mirabilis </italic>
and <italic>Zygeupolia rubens</italic>
.</p>
</sec>
<sec><title>Results</title>
<p>The sizes of the entire mitochondrial genomes are 15365 bp for <italic>N</italic>
. cf. <italic>mirabilis </italic>
and 15513 bp for <italic>Z. rubens</italic>
. 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 <italic>N</italic>
. cf. <italic>mirabilis </italic>
is identical to that of <italic>Paranemertes </italic>
cf. <italic>peregrina</italic>
, both of which are Hoplonemertea, while that of <italic>Z. rubens </italic>
is the same as in <italic>Lineus viridis</italic>
, 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.</p>
</sec>
<sec><title>Conclusion</title>
<p>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.</p>
</sec>
</abstract>
<kwd-group><kwd>MtDNA</kwd>
<kwd>Nemertea</kwd>
<kwd><italic>Nectonemertes mirabilis</italic>
</kwd>
<kwd><italic>Zygeupolia rubens</italic>
</kwd>
<kwd>Phylogeny</kwd>
<kwd>Gene rearrangement</kwd>
</kwd-group>
</article-meta>
</front>
<body><sec><title>Background</title>
<p>Knowledge of mitochondrial genomes is important for many scientific disciplines [<xref ref-type="bibr" rid="B1">1</xref>
,<xref ref-type="bibr" rid="B2">2</xref>
] and the relative arrangement of mitochondrial genes has been effective for studying phylogenetic relationships [<xref ref-type="bibr" rid="B3">3</xref>
,<xref ref-type="bibr" rid="B4">4</xref>
]. However, current knowledge of mtDNAs is uneven, and sequences available in GenBank are predominantly from vertebrate taxa. There are about 1275 described species [<xref ref-type="bibr" rid="B5">5</xref>
] 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, <italic>Cephalothrix hongkongiensis </italic>
(Palaeonemertea) [reported as <italic>Cephalothrix simula </italic>
in [<xref ref-type="bibr" rid="B6">6</xref>
]], <italic>Lineus viridis </italic>
(Heteronemertea) [<xref ref-type="bibr" rid="B7">7</xref>
], and <italic>Paranemertes </italic>
cf. <italic>peregrina </italic>
(Hoplonemertea)[<xref ref-type="bibr" rid="B8">8</xref>
]. Nearly complete sequences exist for the palaeonemerteans <italic>Cephalothrix </italic>
sp. [<xref ref-type="bibr" rid="B8">8</xref>
] and <italic>Cephalothrix rufifrons </italic>
[<xref ref-type="bibr" rid="B9">9</xref>
]. 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, <italic>Nectonemertes </italic>
cf. <italic>mirabilis </italic>
(Hoplonemertea: Polystilifera) and <italic>Zygeupolia rubens </italic>
(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.</p>
</sec>
<sec><title>Results and discussion</title>
<sec><title>Genome organization and structure</title>
<p>Genome composition and gene arrangement of <italic>Nectonemertes </italic>
cf. <italic>mirabilis </italic>
and <italic>Zygeupolia rubens </italic>
are summarized in Figure <xref ref-type="fig" rid="F1">1</xref>
and Table <xref ref-type="table" rid="T1">1</xref>
. The mitochondrial genomes of <italic>N</italic>
. cf. <italic>mirabilis </italic>
and <italic>Z. rubens </italic>
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 <italic>Paranemertes </italic>
cf. <italic>peregrina </italic>
to 16296 bp in <italic>Cephalothrix hongkongiensis </italic>
[<xref ref-type="bibr" rid="B6">6</xref>
]. 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 <italic>trnP </italic>
and <italic>trnT </italic>
are encoded on the same strand (Figure <xref ref-type="fig" rid="F1">1</xref>
).</p>
<fig id="F1" position="float"><label>Figure 1</label>
<caption><p><bold>Circular representation of the mtDNA of <italic>Nectonemertes </italic>
cf. <italic>mirabilis </italic>
and <italic>Zygeupolia rubens</italic>
</bold>
. 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; <italic>trnL1, trnL2, trnS1</italic>
, and <italic>trnS2 </italic>
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. </p>
</caption>
<graphic xlink:href="1471-2164-13-139-1"></graphic>
</fig>
<table-wrap id="T1" position="float"><label>Table 1</label>
<caption><p>Location of genes in the mitochondrial genomes of <italic>Nectonemertes </italic>
cf. <italic>mirabilis </italic>
and <italic>Zygeupolia rubens</italic>
</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th align="left" colspan="4"><italic>Nectonemertes </italic>
cf. <italic>mirabilis</italic>
</th>
<th align="left" colspan="4"><italic>Zygeupolia rubens</italic>
</th>
</tr>
</thead>
<tbody><tr><td align="left"><bold>Genes</bold>
</td>
<td align="left"><bold>From 5'to 3'</bold>
</td>
<td align="left"><bold>Size (bp)</bold>
</td>
<td align="left"><bold>3'spacer</bold>
<sup><bold>a</bold>
</sup>
</td>
<td align="left"><bold>Genes</bold>
</td>
<td align="left"><bold>From 5'to 3'</bold>
</td>
<td align="left"><bold>Size (bp)</bold>
</td>
<td align="left"><bold>3'spacer</bold>
<sup><bold>a</bold>
</sup>
</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnY</italic>
</td>
<td align="left">1-62</td>
<td align="left">62</td>
<td align="left">0</td>
<td align="left"><italic>trnY</italic>
</td>
<td align="left">1-64</td>
<td align="left">64</td>
<td align="left">0</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnP</italic>
<sup>b</sup>
</td>
<td align="left">125-63</td>
<td align="left">63</td>
<td align="left">2</td>
<td align="left"><italic>trnP</italic>
<sup>b</sup>
</td>
<td align="left">131-65</td>
<td align="left">67</td>
<td align="left">3</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>nad6</italic>
</td>
<td align="left">128-583</td>
<td align="left">456</td>
<td align="left">21</td>
<td align="left"><italic>nad6</italic>
</td>
<td align="left">135-599</td>
<td align="left">465</td>
<td align="left">-8</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>cob</italic>
</td>
<td align="left">605-1741</td>
<td align="left">1137</td>
<td align="left">9</td>
<td align="left"><italic>cob</italic>
</td>
<td align="left">592-1728</td>
<td align="left">1137</td>
<td align="left">-1</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnS1 </italic>
(UCN)</td>
<td align="left">1751-1811</td>
<td align="left">61</td>
<td align="left">-1</td>
<td align="left"><italic>trnS1</italic>
(UCN)</td>
<td align="left">1728-1798</td>
<td align="left">71</td>
<td align="left">-1</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnT </italic>
<sup>b</sup>
</td>
<td align="left">1876-1811</td>
<td align="left">66</td>
<td align="left">2</td>
<td align="left"><italic>trnT </italic>
<sup>b</sup>
</td>
<td align="left">1861-1798</td>
<td align="left">64</td>
<td align="left">2</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>nad4L</italic>
</td>
<td align="left">1879-2181</td>
<td align="left">303</td>
<td align="left">-7</td>
<td align="left"><italic>nad4L</italic>
</td>
<td align="left">1864-2169</td>
<td align="left">306</td>
<td align="left">-7</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>nad4</italic>
</td>
<td align="left">2175-3536</td>
<td align="left">1362</td>
<td align="left">6</td>
<td align="left"><italic>nad4</italic>
</td>
<td align="left">2163-3509</td>
<td align="left">1347</td>
<td align="left">1</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnH</italic>
</td>
<td align="left">3543-3602</td>
<td align="left">60</td>
<td align="left">0</td>
<td align="left"><italic>trnH</italic>
</td>
<td align="left">3511-3574</td>
<td align="left">64</td>
<td align="left">2</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>nad5</italic>
</td>
<td align="left">3603-5348</td>
<td align="left">1746</td>
<td align="left">-1</td>
<td align="left"><italic>nad5</italic>
</td>
<td align="left">3577-5308</td>
<td align="left">1732</td>
<td align="left">0</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnE</italic>
</td>
<td align="left">5348-5410</td>
<td align="left">63</td>
<td align="left">1</td>
<td align="left"><italic>trnE</italic>
</td>
<td align="left">5309-5372</td>
<td align="left">64</td>
<td align="left">1</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnG</italic>
</td>
<td align="left">5412-5474</td>
<td align="left">63</td>
<td align="left">2</td>
<td align="left"><italic>trnG</italic>
</td>
<td align="left">5374-5438</td>
<td align="left">65</td>
<td align="left">2</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>cox3</italic>
</td>
<td align="left">5477-6256</td>
<td align="left">780</td>
<td align="left">9</td>
<td align="left"><italic>cox3</italic>
</td>
<td align="left">5441-6220</td>
<td align="left">780</td>
<td align="left">6</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnK</italic>
</td>
<td align="left">6266-6332</td>
<td align="left">67</td>
<td align="left">-2</td>
<td align="left"><italic>trnK</italic>
</td>
<td align="left">6227-6287</td>
<td align="left">61</td>
<td align="left">-1</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnA</italic>
</td>
<td align="left">6331-6393</td>
<td align="left">63</td>
<td align="left">5</td>
<td align="left"><italic>trnA</italic>
</td>
<td align="left">6287-6350</td>
<td align="left">64</td>
<td align="left">0</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnF</italic>
</td>
<td align="left">6399-6464</td>
<td align="left">66</td>
<td align="left">1</td>
<td align="left"><italic>trnF</italic>
</td>
<td align="left">6351-6415</td>
<td align="left">65</td>
<td align="left">0</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnQ</italic>
</td>
<td align="left">6466-6532</td>
<td align="left">67</td>
<td align="left">0</td>
<td align="left"><italic>trnQ</italic>
</td>
<td align="left">6416-6484</td>
<td align="left">69</td>
<td align="left">0</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnR</italic>
</td>
<td align="left">6533-6598</td>
<td align="left">66</td>
<td align="left">1</td>
<td align="left"><italic>trnR</italic>
</td>
<td align="left">6485-6550</td>
<td align="left">66</td>
<td align="left">1</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnN</italic>
</td>
<td align="left">6600-6662</td>
<td align="left">63</td>
<td align="left">2</td>
<td align="left"><italic>trnN</italic>
</td>
<td align="left">6552-6616</td>
<td align="left">65</td>
<td align="left">0</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnI</italic>
</td>
<td align="left">6665-6730</td>
<td align="left">66</td>
<td align="left">1</td>
<td align="left"><italic>trnI</italic>
</td>
<td align="left">6617-6681</td>
<td align="left">65</td>
<td align="left">1</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>nad3</italic>
</td>
<td align="left">6732-7085</td>
<td align="left">354</td>
<td align="left">5</td>
<td align="left"><italic>nad3</italic>
</td>
<td align="left">6683-7039</td>
<td align="left">357</td>
<td align="left">0</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>cox1</italic>
</td>
<td align="left">7091-8626</td>
<td align="left">1536</td>
<td align="left">12</td>
<td align="left">AT-rich</td>
<td align="left">7040-7877</td>
<td align="left">838</td>
<td align="left">0</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnW</italic>
</td>
<td align="left">8639-8703</td>
<td align="left">65</td>
<td align="left">0</td>
<td align="left"><italic>trnS2</italic>
(AGN)</td>
<td align="left">7878-7949</td>
<td align="left">72</td>
<td align="left">0</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left">AT-rich</td>
<td align="left">8704-9405</td>
<td align="left">702</td>
<td align="left">0</td>
<td align="left"><italic>nad2</italic>
</td>
<td align="left">7950-8957</td>
<td align="left">1008</td>
<td align="left">3</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnS2 </italic>
(AGN)</td>
<td align="left">9406-9473</td>
<td align="left">68</td>
<td align="left">-1</td>
<td align="left"><italic>cox1</italic>
</td>
<td align="left">8961-10493</td>
<td align="left">1533</td>
<td align="left">0</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>nad2</italic>
</td>
<td align="left">9473-10480</td>
<td align="left">1008</td>
<td align="left">5</td>
<td align="left"><italic>trnW</italic>
</td>
<td align="left">10494-10558</td>
<td align="left">65</td>
<td align="left">3</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>cox2</italic>
</td>
<td align="left">10486-11166</td>
<td align="left">681</td>
<td align="left">14</td>
<td align="left"><italic>cox2</italic>
</td>
<td align="left">10562-11246</td>
<td align="left">685</td>
<td align="left">0</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnD</italic>
</td>
<td align="left">11181-11245</td>
<td align="left">65</td>
<td align="left">0</td>
<td align="left"><italic>trnD</italic>
</td>
<td align="left">11247-11312</td>
<td align="left">66</td>
<td align="left">0</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>atp8</italic>
</td>
<td align="left">11246-11402</td>
<td align="left">157</td>
<td align="left">40</td>
<td align="left"><italic>atp8</italic>
</td>
<td align="left">11313-11471</td>
<td align="left">159</td>
<td align="left">5</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>atp6</italic>
</td>
<td align="left">11443-12132</td>
<td align="left">700</td>
<td align="left">5</td>
<td align="left"><italic>atp6</italic>
</td>
<td align="left">11477-12169</td>
<td align="left">693</td>
<td align="left">1</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnC</italic>
</td>
<td align="left">12138-12198</td>
<td align="left">61</td>
<td align="left">0</td>
<td align="left"><italic>trnC</italic>
</td>
<td align="left">12171-12232</td>
<td align="left">62</td>
<td align="left">0</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnM</italic>
</td>
<td align="left">12199-12263</td>
<td align="left">65</td>
<td align="left">0</td>
<td align="left"><italic>trnM</italic>
</td>
<td align="left">12233-12296</td>
<td align="left">64</td>
<td align="left">0</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>rrnS</italic>
</td>
<td align="left">12264-13068</td>
<td align="left">805</td>
<td align="left">0</td>
<td align="left"><italic>rrnS</italic>
</td>
<td align="left">12297-13132</td>
<td align="left">836</td>
<td align="left">0</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnV</italic>
</td>
<td align="left">13069-13130</td>
<td align="left">62</td>
<td align="left">0</td>
<td align="left"><italic>trnV</italic>
</td>
<td align="left">13133-13200</td>
<td align="left">68</td>
<td align="left">0</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>rrnL</italic>
</td>
<td align="left">13131-14308</td>
<td align="left">1178</td>
<td align="left">0</td>
<td align="left"><italic>rrnL</italic>
</td>
<td align="left">13201-14448</td>
<td align="left">1248</td>
<td align="left">0</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnL1</italic>
(CUN)</td>
<td align="left">14309-14372</td>
<td align="left">64</td>
<td align="left">1</td>
<td align="left"><italic>trnL1</italic>
(CUN)</td>
<td align="left">14449-14515</td>
<td align="left">67</td>
<td align="left">0</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnL2</italic>
(UUR)</td>
<td align="left">14374-14435</td>
<td align="left">62</td>
<td align="left">2</td>
<td align="left"><italic>trnL2</italic>
(UUR)</td>
<td align="left">14516-14582</td>
<td align="left">67</td>
<td align="left">0</td>
</tr>
<tr><td colspan="8"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>nad1</italic>
</td>
<td align="left">14438-15361</td>
<td align="left">924</td>
<td align="left">4</td>
<td align="left"><italic>nad1</italic>
</td>
<td align="left">14583-15513</td>
<td align="left">931</td>
<td align="left">0</td>
</tr>
</tbody>
</table>
<table-wrap-foot><p><sup>a</sup>
Negative numbers indicate that genes were overlapping</p>
<p><sup>b</sup>
Genes coding in L strand</p>
</table-wrap-foot>
</table-wrap>
<p>For both species, protein-coding genes <italic>nad4L </italic>
and <italic>nad4 </italic>
share an overlap, by seven nucleotides, and <italic>nad6 </italic>
overlaps <italic>cob </italic>
by eight nucleotides in <italic>Z. rubens </italic>
(Figure <xref ref-type="fig" rid="F1">1</xref>
, Table <xref ref-type="table" rid="T1">1</xref>
). Such overlaps are common to all known mtDNA genomes of nemerteans [<xref ref-type="bibr" rid="B6">6</xref>
,<xref ref-type="bibr" rid="B8">8</xref>
], and are found in many metazoan mtDNAs [<xref ref-type="bibr" rid="B10">10</xref>
].</p>
</sec>
<sec><title>Protein-coding genes</title>
<p>Thirteen protein-coding genes (<italic>cox1</italic>
-<italic>cox3, nad1</italic>
-<italic>nad6, nad4L, cob, atp6</italic>
, and <italic>atp8</italic>
) were identified. Mitochondrial genomes often use a variety of nonstandard initiation codons [<xref ref-type="bibr" rid="B11">11</xref>
]. Except for <italic>nad4 </italic>
(GTG), <italic>nad5 </italic>
(GTG), <italic>atp8 </italic>
(GTG) and <italic>atp6 </italic>
(GTT) in <italic>N</italic>
. cf. <italic>mirabilis</italic>
, and <italic>nad1 </italic>
(GTG) and <italic>nad2 </italic>
(GTG) in <italic>Z. rubens</italic>
, 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 (<italic>atp8 </italic>
in <italic>N</italic>
. cf. <italic>mirabilis</italic>
, and <italic>nad5, cox2 </italic>
and <italic>nad1 </italic>
in <italic>Z. rubens</italic>
). Such abbreviated stop codons are common among animal mitochondrial genes. In <italic>Z. rubens</italic>
, the incomplete stop codons are immediately followed by the downstream tRNA gene (Figure <xref ref-type="fig" rid="F1">1</xref>
, Table <xref ref-type="table" rid="T1">1</xref>
), whose secondary structure has been suggested to act as a signal for the cleavage of the polycistronic primary transcript [<xref ref-type="bibr" rid="B12">12</xref>
,<xref ref-type="bibr" rid="B13">13</xref>
]. However, there also are direct junctions pairing ten protein-coding genes in <italic>N</italic>
. cf. <italic>mirabilis </italic>
(<italic>nad6/cob, nad4L</italic>
/<italic>nad4, nad3</italic>
/<italic>cox1, nad2</italic>
/<italic>cox2</italic>
, and <italic>atp8</italic>
/<italic>atp6</italic>
) and eight in <italic>Z. rubens </italic>
(<italic>nad6/cob, nad4L</italic>
/<italic>nad4, nad2</italic>
/<italic>cox1 </italic>
and <italic>atp8</italic>
/<italic>atp6</italic>
) (Figure <xref ref-type="fig" rid="F1">1</xref>
, Table <xref ref-type="table" rid="T1">1</xref>
). Here, cleavage signals other than secondary structure of a tRNA gene may initiate processing of the polycistronic primary transcript [<xref ref-type="bibr" rid="B14">14</xref>
]. For two protein-coding genes (<italic>nad6 </italic>
and <italic>nad2</italic>
) in both nemertean species and <italic>nad3 </italic>
in <italic>N</italic>
. cf. <italic>mirabilis</italic>
, 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 [<xref ref-type="bibr" rid="B15">15</xref>
,<xref ref-type="bibr" rid="B16">16</xref>
].</p>
</sec>
<sec><title>Transfer RNA and ribosomal RNA genes</title>
<p>Both of the mitochondrial genomes encoded 22 tRNA genes found in other nemertean mtDNAs, which is typical of animal mitochondrial genomes [<xref ref-type="bibr" rid="B10">10</xref>
]. They varied from 60 (<italic>trnH</italic>
) to 68 (<italic>trnS2</italic>
) nucleotides in <italic>N</italic>
. cf. <italic>mirabili</italic>
s and 61 (<italic>trnK</italic>
) to 72 (<italic>trnS2</italic>
) nucleotides in <italic>Z. rubens </italic>
(Table <xref ref-type="table" rid="T2">2</xref>
); most were folded into a typical cloverleaf secondary structure (Figures <xref ref-type="fig" rid="F2">2</xref>
, <xref ref-type="fig" rid="F3">3</xref>
). 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., <italic>trnS1 </italic>
of <italic>N</italic>
. cf. <italic>mirabili</italic>
s), as also found in <italic>L. viridis </italic>
and <italic>P</italic>
. cf. <italic>peregrina</italic>
. In general, the lack of a DHU arm in two serine tRNAs is a common condition in metazoan mtDNAs [<xref ref-type="bibr" rid="B17">17</xref>
]. The presence of such aberrant tRNA genes in mitochondrial genomes could be due to modification of tRNA secondary structure by replication slippage [<xref ref-type="bibr" rid="B18">18</xref>
], or selection for mitochondrial genome minimization [<xref ref-type="bibr" rid="B19">19</xref>
].</p>
<table-wrap id="T2" position="float"><label>Table 2</label>
<caption><p>Base composition of the mtDNA in six nemerteans</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th align="left">Species</th>
<th align="left">Total nt</th>
<th align="left">T</th>
<th align="left">C</th>
<th align="left">A</th>
<th align="left">G</th>
<th align="left">A + T</th>
<th align="left">AT skew</th>
<th align="left">GC skew</th>
<th align="left">References</th>
</tr>
</thead>
<tbody><tr><td align="left"><italic>Cephalothrix hongkongiensis</italic>
</td>
<td align="left">16296</td>
<td align="left">47.4</td>
<td align="left">10.2</td>
<td align="left">27.5</td>
<td align="left">14.9</td>
<td align="left">74.9</td>
<td align="left">-0.266</td>
<td align="left">0.187</td>
<td align="left">[<xref ref-type="bibr" rid="B6">6</xref>
]</td>
</tr>
<tr><td colspan="10"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>Cephalothrix </italic>
sp.</td>
<td align="left">15800</td>
<td align="left">47.9</td>
<td align="left">10.0</td>
<td align="left">27.8</td>
<td align="left">14.3</td>
<td align="left">75.7</td>
<td align="left">-0.266</td>
<td align="left">0.178</td>
<td align="left">[<xref ref-type="bibr" rid="B8">8</xref>
]</td>
</tr>
<tr><td colspan="10"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>Paranemertes </italic>
cf. <italic>peregrina</italic>
</td>
<td align="left">14558</td>
<td align="left">47.5</td>
<td align="left">10.0</td>
<td align="left">22.8</td>
<td align="left">19.7</td>
<td align="left">70.3</td>
<td align="left">-0.351</td>
<td align="left">0.322</td>
<td align="left">[<xref ref-type="bibr" rid="B8">8</xref>
]</td>
</tr>
<tr><td colspan="10"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>Nectonemertes </italic>
cf. <italic>mirabilis</italic>
</td>
<td align="left">15365</td>
<td align="left">48.5</td>
<td align="left">10.5</td>
<td align="left">21.8</td>
<td align="left">19.2</td>
<td align="left">70.3</td>
<td align="left">-0.380</td>
<td align="left">0.293</td>
<td align="left">Present study</td>
</tr>
<tr><td colspan="10"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>Lineus viridis</italic>
</td>
<td align="left">15388</td>
<td align="left">44.4</td>
<td align="left">11.9</td>
<td align="left">21.3</td>
<td align="left">22.4</td>
<td align="left">65.7</td>
<td align="left">-0.352</td>
<td align="left">0.306</td>
<td align="left">[<xref ref-type="bibr" rid="B7">7</xref>
]</td>
</tr>
<tr><td colspan="10"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>Zygeupolia rubens</italic>
</td>
<td align="left">15513</td>
<td align="left">45.0</td>
<td align="left">9.8</td>
<td align="left">21.0</td>
<td align="left">24.2</td>
<td align="left">66.0</td>
<td align="left">-0.364</td>
<td align="left">0.424</td>
<td align="left">Present study</td>
</tr>
</tbody>
</table>
</table-wrap>
<fig id="F2" position="float"><label>Figure 2</label>
<caption><p><bold>Secondary structure of tRNA families (<italic>trnA</italic>
-<italic>trnL1</italic>
) in nemertean mtDNAs</bold>
. The nucleotide substitution pattern for each tRNA family was modeled using as reference the structure determined for <italic>Nectonemertes </italic>
cf. <italic>mirabilis</italic>
.</p>
</caption>
<graphic xlink:href="1471-2164-13-139-2"></graphic>
</fig>
<fig id="F3" position="float"><label>Figure 3</label>
<caption><p><bold>Secondary structure of tRNA families (<italic>trnL2</italic>
-<italic>trnV</italic>
) in nemertean mtDNAs</bold>
. The nucleotide substitution pattern for each tRNA family was modeled using as reference the structure determined for <italic>Nectonemertes </italic>
cf. <italic>mirabilis</italic>
.</p>
</caption>
<graphic xlink:href="1471-2164-13-139-3"></graphic>
</fig>
<p>The mtDNAs of nemerteans investigated to date all have 20 tRNAs on the L strand and 2 tRNAs on the H strand ([<xref ref-type="bibr" rid="B6">6</xref>
-<xref ref-type="bibr" rid="B9">9</xref>
]). Secondary structures of nemertean tRNAs are presented and compared in Figures <xref ref-type="fig" rid="F2">2</xref>
and <xref ref-type="fig" rid="F3">3</xref>
(pattern follows [<xref ref-type="bibr" rid="B20">20</xref>
]). Table <xref ref-type="table" rid="T3">3</xref>
presents the tRNA lengths and the percent of identical nucleotides (%INUC) for the six nemerteans.</p>
<table-wrap id="T3" position="float"><label>Table 3</label>
<caption><p>Summary of multiple alignments of tRNA genes in nemertean mtDNAs</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th align="left">ALN</th>
<th align="left">amino acid</th>
<th align="left">alignment<break></break>
length</th>
<th align="left">identical<break></break>
positions</th>
<th align="left">%INUC</th>
</tr>
</thead>
<tbody><tr><td align="left"><italic>trnA</italic>
</td>
<td align="left">Alanine</td>
<td align="left">72</td>
<td align="left">21</td>
<td align="left">29.17</td>
</tr>
<tr><td colspan="5"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnC</italic>
</td>
<td align="left">Cysteine</td>
<td align="left">66</td>
<td align="left">39</td>
<td align="left">59.09</td>
</tr>
<tr><td colspan="5"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnD</italic>
</td>
<td align="left">Aspartate</td>
<td align="left">66</td>
<td align="left">26</td>
<td align="left">39.39</td>
</tr>
<tr><td colspan="5"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnE</italic>
</td>
<td align="left">Glutamate</td>
<td align="left">65</td>
<td align="left">27</td>
<td align="left">41.54</td>
</tr>
<tr><td colspan="5"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnF</italic>
</td>
<td align="left">Phenylalanine</td>
<td align="left">68</td>
<td align="left">22</td>
<td align="left">32.35</td>
</tr>
<tr><td colspan="5"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnG</italic>
</td>
<td align="left">Glycine</td>
<td align="left">67</td>
<td align="left">39</td>
<td align="left">58.21</td>
</tr>
<tr><td colspan="5"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnH</italic>
</td>
<td align="left">Histidine</td>
<td align="left">67</td>
<td align="left">25</td>
<td align="left">37.31</td>
</tr>
<tr><td colspan="5"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnI</italic>
</td>
<td align="left">Isoleucine</td>
<td align="left">72</td>
<td align="left">28</td>
<td align="left">38.89</td>
</tr>
<tr><td colspan="5"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnK</italic>
</td>
<td align="left">Lysine</td>
<td align="left">73</td>
<td align="left">23</td>
<td align="left">31.51</td>
</tr>
<tr><td colspan="5"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnL1</italic>
</td>
<td align="left">Leucine (CUN)</td>
<td align="left">69</td>
<td align="left">21</td>
<td align="left">30.43</td>
</tr>
<tr><td colspan="5"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnL2</italic>
</td>
<td align="left">Leucine (UUR)</td>
<td align="left">68</td>
<td align="left">28</td>
<td align="left">41.18</td>
</tr>
<tr><td colspan="5"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnM</italic>
</td>
<td align="left">Methionine</td>
<td align="left">66</td>
<td align="left">38</td>
<td align="left">57.58</td>
</tr>
<tr><td colspan="5"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnN</italic>
</td>
<td align="left">Asparagine</td>
<td align="left">69</td>
<td align="left">20</td>
<td align="left">28.99</td>
</tr>
<tr><td colspan="5"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnP<sup>a</sup>
</italic>
</td>
<td align="left">Proline</td>
<td align="left">67</td>
<td align="left">24</td>
<td align="left">35.82</td>
</tr>
<tr><td colspan="5"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnQ</italic>
</td>
<td align="left">Glutamine</td>
<td align="left">70</td>
<td align="left">28</td>
<td align="left">40.00</td>
</tr>
<tr><td colspan="5"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnR</italic>
</td>
<td align="left">Arginine</td>
<td align="left">67</td>
<td align="left">16</td>
<td align="left">23.88</td>
</tr>
<tr><td colspan="5"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnS1</italic>
</td>
<td align="left">Serine (UCN)</td>
<td align="left">71</td>
<td align="left">23</td>
<td align="left">32.39</td>
</tr>
<tr><td colspan="5"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnS2</italic>
</td>
<td align="left">Serine (AGN)</td>
<td align="left">73</td>
<td align="left">30</td>
<td align="left">41.10</td>
</tr>
<tr><td colspan="5"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnT<sup>a</sup>
</italic>
</td>
<td align="left">Threonine</td>
<td align="left">71</td>
<td align="left">23</td>
<td align="left">32.39</td>
</tr>
<tr><td colspan="5"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnV</italic>
</td>
<td align="left">Valine</td>
<td align="left">69</td>
<td align="left">33</td>
<td align="left">47.83</td>
</tr>
<tr><td colspan="5"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnW</italic>
</td>
<td align="left">Tryptophan</td>
<td align="left">70</td>
<td align="left">27</td>
<td align="left">38.57</td>
</tr>
<tr><td colspan="5"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>trnY</italic>
</td>
<td align="left">Tyrosine</td>
<td align="left">68</td>
<td align="left">32</td>
<td align="left">47.06</td>
</tr>
</tbody>
</table>
<table-wrap-foot><p>ALN, alignment name; %INUC, percent of identical nucleotides</p>
<p><sup>a</sup>
genes on the L strand</p>
</table-wrap-foot>
</table-wrap>
<p>Nucleotide conservation was strongest on the H strand, with <italic>trnC, trnG </italic>
and <italic>trnM</italic>
, having the highest levels of nucleotide conservation (%INUC > 50), followed by <italic>TrnE, trnL2, trnQ, trnS2, trnV </italic>
and <italic>trnY </italic>
at 40 ≤ %INUC ≤ 50 (Figure <xref ref-type="fig" rid="F2">2</xref>
). The ten remaining tRNAs had %INUC values between 30 and 40; eight - <italic>trnD, trnF, trnH, trnI, trnK, trnL1, trnS1 </italic>
and <italic>trnW </italic>
- are located on the H strand, while two - <italic>trnP </italic>
and <italic>trnT </italic>
- are on the L strand. H-strand genes <italic>trnA, trnN </italic>
and <italic>trnR </italic>
had %INUC values ≤30.</p>
<p>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, <italic>trnC </italic>
and <italic>trnM</italic>
, are adjoining, while the third, <italic>trnG</italic>
, adjoins the moderately conserved <italic>trnE </italic>
and is relatively close to the three least conserved genes, <italic>trnA, trnN </italic>
and <italic>trnR </italic>
(Figure <xref ref-type="fig" rid="F1">1</xref>
, Table <xref ref-type="table" rid="T1">1</xref>
). As observed by others (e.g., [<xref ref-type="bibr" rid="B20">20</xref>
]), 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).</p>
<p>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. [<xref ref-type="bibr" rid="B20">20</xref>
] for arthropods, metazoan mtDNAs commonly have such mismatches. It has been suggested that these may be corrected via RNA-editing mechanisms (e.g., [<xref ref-type="bibr" rid="B17">17</xref>
]) or they may represent unusual pairings [<xref ref-type="bibr" rid="B21">21</xref>
].</p>
<p>Among the most conserved tRNAs in nemerteans, as in insects (e.g., [<xref ref-type="bibr" rid="B20">20</xref>
]), nucleotide substitutions are mostly confined to TΨC and DHU loops and extra arms (Figures <xref ref-type="fig" rid="F2">2</xref>
, <xref ref-type="fig" rid="F3">3</xref>
), 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 [<xref ref-type="bibr" rid="B20">20</xref>
,<xref ref-type="bibr" rid="B22">22</xref>
]). As in insects [<xref ref-type="bibr" rid="B20">20</xref>
], the number of cbcs and hemi-cbcs increased in stems as overall variation increased, especially in the TΨC stem.</p>
<p>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 <italic>trnC </italic>
acceptor arm of <italic>P</italic>
. cf. <italic>peregrina</italic>
, which was mirrored by G-C in all other nemerteans (Figure <xref ref-type="fig" rid="F2">2</xref>
). Few substitutions were present among <italic>C. hongkongiensis </italic>
and <italic>Cephalothrix </italic>
sp. (Figures <xref ref-type="fig" rid="F2">2</xref>
, <xref ref-type="fig" rid="F3">3</xref>
). An example of a full cbc characterizing a unique family is the A-T pair found in the acceptor stem of <italic>trnL1s </italic>
of family Lineidae (<italic>L. viridis </italic>
and <italic>Z. rubens</italic>
), while other taxa exhibited the G-C pair (Figure <xref ref-type="fig" rid="F2">2</xref>
). Similarly, a full cbc in the anticodon stem of <italic>trnG </italic>
of two hoplonemerteans characterizes another high-taxonomic rank (Figure <xref ref-type="fig" rid="F2">2</xref>
). Figures <xref ref-type="fig" rid="F2">2</xref>
and <xref ref-type="fig" rid="F3">3</xref>
depict several more examples. This points to the potential phylogenetic value of tRNA sequences, as demonstrated for other animal groups (e.g., [<xref ref-type="bibr" rid="B20">20</xref>
,<xref ref-type="bibr" rid="B23">23</xref>
]), especially when secondary structures are taken into account [<xref ref-type="bibr" rid="B20">20</xref>
]. While encouraging, clearly we need substantially more nemertean mitochondrial genomes to test this assertion for nemerteans.</p>
<p>The anticodon usage of <italic>N</italic>
. cf. <italic>mirabili</italic>
s and <italic>Z. rubens </italic>
was congruent with the corresponding tRNA genes of other nemerteans, with one exception. The anticodon of the <italic>trnS2 </italic>
(AGN) gene in <italic>N</italic>
. cf. <italic>mirabili</italic>
s, <italic>P</italic>
. cf. <italic>peregrina </italic>
and three <italic>Cephalothrix </italic>
species is GCT, but it is TCT in <italic>L. viridis </italic>
and <italic>Z. rubens</italic>
. Cameron et al. [<xref ref-type="bibr" rid="B24">24</xref>
] found that anticodon changes in <italic>trnS2 </italic>
(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" [<xref ref-type="bibr" rid="B25">25</xref>
] in nemerteans and be a synapomorphy of Lineidae.</p>
<p>As in all other metazoan mtDNAs sequenced to date, <italic>N</italic>
. cf. <italic>mirabili</italic>
s and <italic>Z. rubens </italic>
mtDNAs contain genes for both small and large ribosomal subunit RNAs (<italic>rrnS </italic>
and <italic>rrnL</italic>
). Both genes are encoded by the same strand and are separated by <italic>trnV</italic>
, as in many other metazoans. For <italic>N</italic>
. cf. <italic>mirabili</italic>
s and <italic>Z. rubens</italic>
, respectively, the lengths of <italic>rrnL</italic>
/<italic>rrnS </italic>
are 1178/805 bp and 1248/836 bp, and the A + T contents are 75.5/72.4% and 70.9/70.5%.</p>
</sec>
<sec><title>Base composition and codon usage</title>
<p>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 <italic>N</italic>
. cf. <italic>mirabili</italic>
s and <italic>Z. rubens </italic>
(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., <italic>Cephalothrix </italic>
sp./<italic>C. hongkongiensis</italic>
), as well as in more inclusive taxa (e.g., order - e.g., <italic>P</italic>
. cf. <italic>peregrina</italic>
/<italic>N</italic>
. cf. <italic>mirabili</italic>
s; <italic>L. viridis/Z. rubens</italic>
) (Table <xref ref-type="table" rid="T2">2</xref>
). This might indicate a phylogenetic signal in nemerteans.</p>
<p>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 [<xref ref-type="bibr" rid="B26">26</xref>
]. 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 <xref ref-type="table" rid="T2">2</xref>
; Figure <xref ref-type="fig" rid="F4">4</xref>
). This situation is common for mitochondrial genomes [<xref ref-type="bibr" rid="B26">26</xref>
] and may be due to the presence of asymmetric patterns of mutational changes between strands [<xref ref-type="bibr" rid="B27">27</xref>
,<xref ref-type="bibr" rid="B28">28</xref>
], and has been related with nucleotide deamination of DNA while transiently single-stranded during replication (this is not without controversy [<xref ref-type="bibr" rid="B29">29</xref>
]) and/or transcription [<xref ref-type="bibr" rid="B30">30</xref>
]. The relative importance of the two contributing processes (i.e., transcription vs. replication) remains to be assessed.</p>
<fig id="F4" position="float"><label>Figure 4</label>
<caption><p><bold>Graphical representation of the percentage of A (black) and T (gray) across the whole mtDNA segment of six nemertean species (Accelrys)</bold>
. Y-axis values represent nucleotide %, calculated with a 100-bp sliding window using the program MacVector<sup>® </sup>
7.2.3; x-axis values represent the nucleotide positions corresponding to the linearized genome.</p>
</caption>
<graphic xlink:href="1471-2164-13-139-4"></graphic>
</fig>
<p>We follow the pattern of [<xref ref-type="bibr" rid="B2">2</xref>
] for displaying codon family abundance and relative synonymous codon usage (RSCU) for available nemertean protein-coding genes (Figures <xref ref-type="fig" rid="F5">5</xref>
and <xref ref-type="fig" rid="F6">6</xref>
). 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 <italic>P</italic>
. cf. <italic>peregrina </italic>
to 3707 in <italic>L. viridis</italic>
. 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 <xref ref-type="fig" rid="F5">5</xref>
), with CDs rich in A + T favored over synonymous CDs of lower A + T content (Figure <xref ref-type="fig" rid="F6">6</xref>
). 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.</p>
<fig id="F5" position="float"><label>Figure 5</label>
<caption><p><bold>Codon distribution in nemertean mtDNAs</bold>
. CDspT, number of codons per thousands codons. Numbers to the right refer to the total number of codons.</p>
</caption>
<graphic xlink:href="1471-2164-13-139-5"></graphic>
</fig>
<fig id="F6" position="float"><label>Figure 6</label>
<caption><p><bold>Relative Synonymous Codon Usage (RSCU) in nemertean mtDNAs</bold>
. Codon families are provided on the x-axis, codons not present in the genome are orange colored.</p>
</caption>
<graphic xlink:href="1471-2164-13-139-6"></graphic>
</fig>
<p>The invertebrate mitochondrial genome codes for 62 amino-acid codons [<xref ref-type="bibr" rid="B10">10</xref>
]. As pointed out for Lepidoptera [<xref ref-type="bibr" rid="B2">2</xref>
], 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, <italic>Cephalothrix </italic>
sp. mtDNA has the highest (A + T)% content (see Table <xref ref-type="table" rid="T2">2</xref>
) and uses only 58 codons, never using the four codons rich in G + C (TCG, CGC, ACG, CGC) (Figure <xref ref-type="fig" rid="F6">6</xref>
). <italic>Lineus viridis </italic>
mtDNA uses all 62 codons and has the lowest A + T% among known nemertean genomes.</p>
<p>The abundance of the four amino acid residues - Leu, Ile, Phe and Ser - is typical for invertebrate membrane proteins [<xref ref-type="bibr" rid="B2">2</xref>
,<xref ref-type="bibr" rid="B31">31</xref>
], 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.</p>
<p>Two- and four-fold degenerate codon usage was similarly biased, with A/T favored over G/C in the third position (Figure <xref ref-type="fig" rid="F6">6</xref>
) and in agreement with the AT-bias of protein-coding genes. Since the nemertean mitochondrial genome is AT-rich (Table <xref ref-type="table" rid="T2">2</xref>
), 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.</p>
</sec>
<sec><title>Non-coding regions</title>
<p>Metazoan mtDNAs usually have lengthy non-coding regions varying in size from ~100 bp to > 20 kbp [<xref ref-type="bibr" rid="B32">32</xref>
,<xref ref-type="bibr" rid="B33">33</xref>
]. The mtDNAs of <italic>N</italic>
. cf. <italic>mirabili</italic>
s and <italic>Z. rubens </italic>
contain a large number of unassigned nucleotides. There are 23 non-coding regions, with up to 855 nts, found throughout the <italic>N</italic>
. cf. <italic>mirabili</italic>
s mitochondrial genome. The AT-rich region located between the <italic>nad3 </italic>
and <italic>trnS2 </italic>
genes accounts for 838 nts and its AT content is 81.5%, which is higher than the remainder of the genome. <italic>Zygeupolia rubens </italic>
has up to 879 non-coding nts distributed in 15 regions. The AT-rich region located between <italic>trnW </italic>
and <italic>trnS2 </italic>
genes is 702 nts and has an AT content of 74.9%, which also is higher than the remainder of the genome.</p>
<p>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 [<xref ref-type="bibr" rid="B11">11</xref>
]. The non-coding region has an increased AT composition, a characteristic typically used to identify origins of replication [<xref ref-type="bibr" rid="B10">10</xref>
]. As in mtDNA genomes of other nemerteans, the AT-rich regions of <italic>N</italic>
. cf. <italic>mirabili</italic>
s and <italic>Z. rubens </italic>
mtDNAs have the potential to form secondary structures such as stems and loops (Figure <xref ref-type="fig" rid="F7">7</xref>
), which are thought to play an important role in the early stages of the replication and transcription process [<xref ref-type="bibr" rid="B34">34</xref>
,<xref ref-type="bibr" rid="B35">35</xref>
]. Additionally, the AT-rich region in mtDNA of <italic>N</italic>
. cf. <italic>mirabili</italic>
s contains the tandemly repeated sequences (AAAAATATAAGATTTTTCAAATTCCAAAAATATAAAAT)<sub>3</sub>
, (TTTTG)<sub>10</sub>
, (TTTTTC)<sub>7</sub>
, and several (A)<sub>n </sub>
and (T)<sub>n </sub>
homopolymer tracts. In mtDNAs of <italic>Z. rubens</italic>
, we found the tandemly repeated sequences (GGGGGGGGGGGTAGTGTGGTTATGTTTTACTACACTCTTAGTAAAATATAAA)<sub>2</sub>
, (TTTTTTG)<sub>10</sub>
, and (TTTTTTTTA)<sub>6</sub>
. Similar tandem repeat units within the largest non-coding regions also were found in the nemerteans <italic>Cephalothrix </italic>
sp. [<xref ref-type="bibr" rid="B8">8</xref>
], and <italic>C. hongkongiensis </italic>
[<xref ref-type="bibr" rid="B6">6</xref>
]. Tandem repeats are common within the control region of animal mtDNAs [<xref ref-type="bibr" rid="B34">34</xref>
] and might be associated with regulatory mechanisms and recombination hot spots, and they might be the result of replication slippage events [<xref ref-type="bibr" rid="B36">36</xref>
]. The high AT content and the predicted secondary structures of the AT-rich non-coding region of the <italic>N</italic>
. cf. <italic>mirabili</italic>
s and <italic>Z. rubens </italic>
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 [<xref ref-type="bibr" rid="B37">37</xref>
]. 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., [<xref ref-type="bibr" rid="B6">6</xref>
,<xref ref-type="bibr" rid="B8">8</xref>
]), nucleotide identities or potential secondary structures for the nemertean non-coding regions.</p>
<fig id="F7" position="float"><label>Figure 7</label>
<caption><p><bold>Secondary structures predicted for the non-coding regions in the mitochondrial genome of two nemerteans</bold>
. (A) <italic>Nectonemertes </italic>
cf. <italic>mirabilis</italic>
, AT-rich non-coding region between genes <italic>trnW </italic>
and <italic>trnS2</italic>
; (B, C) <italic>Zygeupolia rubens</italic>
, AT-rich non-coding region between genes <italic>nad3 </italic>
and <italic>trnS2</italic>
.</p>
</caption>
<graphic xlink:href="1471-2164-13-139-7"></graphic>
</fig>
</sec>
<sec><title>Gene order comparison</title>
<p>Gene arrangements of the animal mitochondrial genome usually remain stable over long periods of evolutionary time, especially for protein-coding genes [<xref ref-type="bibr" rid="B10">10</xref>
]. With some exceptions, mitochondrial gene order is relatively stable within major groups, and more variable between them [<xref ref-type="bibr" rid="B14">14</xref>
]. This is the case for available nemertean mtDNA genomes, with mitochondrial genes transcribed from the same strand except for <italic>trnP </italic>
and <italic>trnT</italic>
. Among the three species of <italic>Cephalothrix </italic>
(<italic>C. hongkongiensis, C</italic>
. sp. and <italic>C. rufifrons</italic>
), the gene order is identical for two but that of <italic>C. rufifrons </italic>
differs from them. The two hoplonemertean species (<italic>P</italic>
. cf. <italic>peregrina, N</italic>
. cf. <italic>mirabili</italic>
s) are identical to each other in gene order, as is the case for the two heteronemerteans (<italic>Z. rubens, L. viridis</italic>
). The hoplo- and the heteronemertean species differ only by a translocation of the gene block S2/<italic>nad2 </italic>
but they differ significantly from the three <italic>Cephalothrix </italic>
species in the positions of <italic>atp8, nad6, nad2 </italic>
and several tRNAs. The highest number of common intervals (1124) is between hoplo- and heteronemerteans, as indicated by results from CREx [<xref ref-type="bibr" rid="B38">38</xref>
].</p>
<p>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 [<xref ref-type="bibr" rid="B39">39</xref>
] and to mitochondrial gene orders of various lophotrochozoans: <italic>Terebratulina retusa </italic>
(Brachiopoda) [<xref ref-type="bibr" rid="B40">40</xref>
], <italic>Katharina tunicata </italic>
(Mollusca)[<xref ref-type="bibr" rid="B14">14</xref>
], <italic>Phoronis psammophila </italic>
(Phoronida) [<xref ref-type="bibr" rid="B41">41</xref>
], <italic>Perionyx excavatus </italic>
(Annelida) [<xref ref-type="bibr" rid="B42">42</xref>
], <italic>Urechis caupo </italic>
(Annelida) [<xref ref-type="bibr" rid="B43">43</xref>
] and <italic>Sipunculus nudus </italic>
(Annelida)[<xref ref-type="bibr" rid="B44">44</xref>
]. For the "all genes" set, all nemerteans share the adjacency <italic>nad4L/nad4 </italic>
with the ground pattern of Bilateria and with the selected species (Figure <xref ref-type="fig" rid="F8">8</xref>
). Nemerteans share the adjacencies <italic>rrnS</italic>
/V/<italic>rrnL </italic>
with Bilateria and the other species except <italic>U. caupo</italic>
. The adjacency H/<italic>nad5 </italic>
is shared by nemerteans and the selected species. Based on both gene sets, the hetero- and hoplonemerteans share the adjacency <italic>nad6/cob </italic>
with <italic>K. tunicata </italic>
[<xref ref-type="bibr" rid="B14">14</xref>
], <italic>P. psammophila </italic>
[<xref ref-type="bibr" rid="B41">41</xref>
], <italic>P. excavatus </italic>
[<xref ref-type="bibr" rid="B42">42</xref>
], <italic>U. caupo </italic>
[<xref ref-type="bibr" rid="B43">43</xref>
], and <italic>S. nudus </italic>
[<xref ref-type="bibr" rid="B44">44</xref>
] and they share the adjacency <italic>atp8/atp6 </italic>
with <italic>T. retusa, K. tunicata </italic>
and the putative ground pattern of Bilateria (Figure <xref ref-type="fig" rid="F8">8</xref>
; Additional file <xref ref-type="supplementary-material" rid="S1">1</xref>
: Figure S1). These adjacencies may be a common plesiomorphic feature of Lophotrochozoa, such as Mollusca, Brachiopoda, and also Arthropoda mitochondrial genomes (e.g., [<xref ref-type="bibr" rid="B10">10</xref>
]; [<xref ref-type="bibr" rid="B44">44</xref>
]). However, neither of the latter two adjacencies is present in two <italic>Cephalothrix </italic>
species, nor in the bryozoan <italic>Flustrellidra hispida </italic>
[<xref ref-type="bibr" rid="B45">45</xref>
].</p>
<fig id="F8" position="float"><label>Figure 8</label>
<caption><p><bold>Mitochondrial gene order (all 37 genes) of Nemertea and selected lophotrochozoan species and the putative bilaterian ground pattern (according to </bold>
[<xref ref-type="bibr" rid="B39">39</xref>
]<bold>)</bold>
. 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: <italic>Cephalothrix </italic>
[<xref ref-type="bibr" rid="B6">6</xref>
,<xref ref-type="bibr" rid="B8">8</xref>
], <italic>Lineus </italic>
[<xref ref-type="bibr" rid="B7">7</xref>
], <italic>Paranemertes </italic>
[<xref ref-type="bibr" rid="B8">8</xref>
], <italic>Terebratulina </italic>
[<xref ref-type="bibr" rid="B40">40</xref>
], <italic>Katharina </italic>
[<xref ref-type="bibr" rid="B14">14</xref>
], <italic>Phoronis </italic>
[<xref ref-type="bibr" rid="B41">41</xref>
], <italic>Perionyx </italic>
[<xref ref-type="bibr" rid="B42">42</xref>
], <italic>Urechis </italic>
[<xref ref-type="bibr" rid="B43">43</xref>
], <italic>Sipunculus </italic>
[<xref ref-type="bibr" rid="B44">44</xref>
].</p>
</caption>
<graphic xlink:href="1471-2164-13-139-8"></graphic>
</fig>
<p>In addition to visual comparison of genome maps, we analyzed gene order data with CREx [<xref ref-type="bibr" rid="B38">38</xref>
], determining the number of common intervals. As shown in Table <xref ref-type="table" rid="T4">4</xref>
, the nemerteans share the highest number of common intervals (154, 184, 212) with <italic>K. tunicata </italic>
and with <italic>P. psammophila </italic>
(but this is a partial mitochondrial genome), while the lowest number was obtained in comparison with <italic>U. caupo </italic>
(28, 18, 18). Although not significant, nemerteans and <italic>T. retusa, K. tunicata</italic>
, and <italic>P. excavatus </italic>
yield the highest numbers (18-20) in comparison with the putative bilaterian ground pattern.</p>
<table-wrap id="T4" position="float"><label>Table 4</label>
<caption><p>Pairwise common interval distance matrix of mitochondrial gene orders of nemerteans, the putative bilaterian ground pattern and six other lophotrochozoans *</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th align="left">Common interval</th>
<th align="left">B</th>
<th align="left">P</th>
<th align="left">H</th>
<th align="left">H</th>
<th align="left">Tr</th>
<th align="left">Kt</th>
<th align="left">Uc</th>
<th align="left">Sn</th>
<th align="left">Pe</th>
<th align="left">Pp</th>
</tr>
</thead>
<tbody><tr><td align="left">Bilaterian ground pattern (B)</td>
<td align="left">204\<bold>1326</bold>
</td>
<td align="left"><bold>18</bold>
</td>
<td align="left"><bold>20</bold>
</td>
<td align="left"><bold>20</bold>
</td>
<td align="left"><bold>18</bold>
</td>
<td align="left"><bold>20</bold>
</td>
<td align="left"><bold>12</bold>
</td>
<td align="left"><bold>14</bold>
</td>
<td align="left"><bold>20</bold>
</td>
<td align="left"><bold>12</bold>
</td>
</tr>
<tr><td colspan="11"><hr></hr>
</td>
</tr>
<tr><td align="left">Palaeonemertean (P)</td>
<td align="left"><italic>44</italic>
</td>
<td align="left">204\<bold>1326</bold>
</td>
<td align="left"><bold>108</bold>
</td>
<td align="left"><bold>112</bold>
</td>
<td align="left"><bold>40</bold>
</td>
<td align="left"><bold>154</bold>
</td>
<td align="left"><bold>28</bold>
</td>
<td align="left"><bold>42</bold>
</td>
<td align="left"><bold>38</bold>
</td>
<td align="left"><bold>142</bold>
</td>
</tr>
<tr><td colspan="11"><hr></hr>
</td>
</tr>
<tr><td align="left">Heteronemertean (H)</td>
<td align="left"><italic>52</italic>
</td>
<td align="left"><italic>86</italic>
</td>
<td align="left">204\<bold>1326</bold>
</td>
<td align="left"><bold>1124</bold>
</td>
<td align="left"><bold>68</bold>
</td>
<td align="left"><bold>184</bold>
</td>
<td align="left"><bold>18</bold>
</td>
<td align="left"><bold>64</bold>
</td>
<td align="left"><bold>56</bold>
</td>
<td align="left"><bold>230</bold>
</td>
</tr>
<tr><td colspan="11"><hr></hr>
</td>
</tr>
<tr><td align="left">Hoplonemertean (H)</td>
<td align="left"><italic>44</italic>
</td>
<td align="left"><italic>72</italic>
</td>
<td align="left"><italic>178</italic>
</td>
<td align="left">204\<bold>1326</bold>
</td>
<td align="left"><bold>84</bold>
</td>
<td align="left"><bold>212</bold>
</td>
<td align="left"><bold>18</bold>
</td>
<td align="left"><bold>68</bold>
</td>
<td align="left"><bold>66</bold>
</td>
<td align="left"><bold>254</bold>
</td>
</tr>
<tr><td colspan="11"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>Terebratulina retusa </italic>
(Tr)</td>
<td align="left"><italic>52</italic>
</td>
<td align="left"><italic>86</italic>
</td>
<td align="left"><italic>204</italic>
</td>
<td align="left"><italic>178</italic>
</td>
<td align="left">204\<bold>1326</bold>
</td>
<td align="left"><bold>128</bold>
</td>
<td align="left"><bold>20</bold>
</td>
<td align="left"><bold>74</bold>
</td>
<td align="left"><bold>82</bold>
</td>
<td align="left"><bold>110</bold>
</td>
</tr>
<tr><td colspan="11"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>Katharina tunicata </italic>
(Kt)</td>
<td align="left"><italic>48</italic>
</td>
<td align="left"><italic>56</italic>
</td>
<td align="left"><italic>106</italic>
</td>
<td align="left"><italic>94</italic>
</td>
<td align="left"><italic>106</italic>
</td>
<td align="left">204\<bold>1326</bold>
</td>
<td align="left"><bold>20</bold>
</td>
<td align="left"><bold>62</bold>
</td>
<td align="left"><bold>64</bold>
</td>
<td align="left"><bold>266</bold>
</td>
</tr>
<tr><td colspan="11"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>Urechis caupo </italic>
(Uc)</td>
<td align="left"><italic>16</italic>
</td>
<td align="left"><italic>8</italic>
</td>
<td align="left"><italic>14</italic>
</td>
<td align="left"><italic>8</italic>
</td>
<td align="left"><italic>14</italic>
</td>
<td align="left"><italic>34</italic>
</td>
<td align="left">204\<bold>1326</bold>
</td>
<td align="left"><bold>54</bold>
</td>
<td align="left"><bold>144</bold>
</td>
<td align="left"><bold>22</bold>
</td>
</tr>
<tr><td colspan="11"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>Sipunculus nudus </italic>
(Sn)</td>
<td align="left"><italic>34</italic>
</td>
<td align="left"><italic>12</italic>
</td>
<td align="left"><italic>22</italic>
</td>
<td align="left"><italic>16</italic>
</td>
<td align="left"><italic>22</italic>
</td>
<td align="left"><italic>26</italic>
</td>
<td align="left"><italic>26</italic>
</td>
<td align="left">204\<bold>1326</bold>
</td>
<td align="left"><bold>158</bold>
</td>
<td align="left"><bold>38</bold>
</td>
</tr>
<tr><td colspan="11"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>Perionyx excavatus </italic>
(Pe)<sup>a</sup>
</td>
<td align="left"><italic>28</italic>
</td>
<td align="left"><italic>24</italic>
</td>
<td align="left"><italic>40</italic>
</td>
<td align="left"><italic>32</italic>
</td>
<td align="left"><italic>40</italic>
</td>
<td align="left"><italic>48</italic>
</td>
<td align="left"><italic>44</italic>
</td>
<td align="left"><italic>60</italic>
</td>
<td align="left">204\<bold>1254</bold>
</td>
<td align="left"><bold>44</bold>
</td>
</tr>
<tr><td colspan="11"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>Phoronis psammophila </italic>
(Pp)<sup>b</sup>
</td>
<td align="left"><italic>40</italic>
</td>
<td align="left"><italic>48</italic>
</td>
<td align="left"><italic>84</italic>
</td>
<td align="left"><italic>76</italic>
</td>
<td align="left"><italic>84</italic>
</td>
<td align="left"><italic>98</italic>
</td>
<td align="left"><italic>22</italic>
</td>
<td align="left"><italic>24</italic>
</td>
<td align="left"><italic>38</italic>
</td>
<td align="left">204\<bold>864</bold>
</td>
</tr>
</tbody>
</table>
<table-wrap-foot><p>*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)</p>
<p><sup>a</sup>
lacks <italic>trnR</italic>
</p>
<p><sup>b</sup>
lacks several tRNAs</p>
</table-wrap-foot>
</table-wrap>
<p>Figure <xref ref-type="fig" rid="F8">8</xref>
shows tRNA genes change relative position much faster than the protein-coding and rRNA genes, as reported from previous studies (e.g., [<xref ref-type="bibr" rid="B46">46</xref>
,<xref ref-type="bibr" rid="B47">47</xref>
]).</p>
<p>Excluding tRNAs, the gene order of heteronemerteans is identical to that of <italic>T. retusa </italic>
[<xref ref-type="bibr" rid="B40">40</xref>
] and some gastropods, e.g., <italic>Conus textile </italic>
[<xref ref-type="bibr" rid="B48">48</xref>
], <italic>Ilyanassa obsoleta </italic>
[<xref ref-type="bibr" rid="B49">49</xref>
], <italic>Thais clavigera </italic>
[<xref ref-type="bibr" rid="B37">37</xref>
] and <italic>Lophiotoma cerithiformis </italic>
[<xref ref-type="bibr" rid="B50">50</xref>
]. Other molluscs, like the polyplacophoran <italic>K. tunicata </italic>
[<xref ref-type="bibr" rid="B14">14</xref>
], the gastropod <italic>Haliotis rubra </italic>
[<xref ref-type="bibr" rid="B51">51</xref>
] and the cephalopod <italic>Octopus vulgaris </italic>
[<xref ref-type="bibr" rid="B52">52</xref>
] show a similar gene order, but are distinguished by a large inversion of about half the mt genome (Additional file <xref ref-type="supplementary-material" rid="S1">1</xref>
: Figure S1). Without tRNAs, heteronemerteans and <italic>T. retusa</italic>
, which has the same gene order, share the greatest number of possible common intervals (204) (Table <xref ref-type="table" rid="T4">4</xref>
), and both share the greatest number (52) with the putative bilaterian ground pattern.</p>
<p>We also determined breakpoints and reversal distances between these taxa with the two gene sets (Additional files <xref ref-type="supplementary-material" rid="S2">2</xref>
, <xref ref-type="supplementary-material" rid="S3">3</xref>
: 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 [<xref ref-type="bibr" rid="B7">7</xref>
].</p>
</sec>
<sec><title>Phylogenetic analysis</title>
<p>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 <xref ref-type="fig" rid="F9">9</xref>
, 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., [<xref ref-type="bibr" rid="B53">53</xref>
-<xref ref-type="bibr" rid="B55">55</xref>
]). 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., [<xref ref-type="bibr" rid="B55">55</xref>
]). 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.</p>
<fig id="F9" position="float"><label>Figure 9</label>
<caption><p><bold>Best tree from the Maximum Likelihood analysis with 5921 nt (first and second codon positions) of protein-coding genes</bold>
. 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.</p>
</caption>
<graphic xlink:href="1471-2164-13-139-9"></graphic>
</fig>
</sec>
</sec>
<sec sec-type="conclusions"><title>Conclusion</title>
<p>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 <italic>Nectonemertes </italic>
cf. <italic>mirabilis </italic>
and <italic>Zygeupolia rubens</italic>
, 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.</p>
<p>There is strong skew in the distribution of nucleotides between the two strands.</p>
<p>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.</p>
<p>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.</p>
<p>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.</p>
</sec>
<sec sec-type="methods"><title>Methods</title>
<sec><title>DNA extraction, PCR and sequencing</title>
<p>Specimens were collected off Point Conception, California (<italic>Nectonemertes </italic>
cf. <italic>mirabilis</italic>
) and at Fort Pierce, Florida (<italic>Zygeupolia rubens</italic>
), USA. We use the "cf." qualifier to confer reasonable caution that the Pacific worm traditionally designated <italic>N. mirabilis </italic>
(see [<xref ref-type="bibr" rid="B56">56</xref>
]) 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 <xref ref-type="table" rid="T5">5</xref>
. Preliminary nemertean-specific primers (N12SF, N16SR, NCOX2R) were designed based on sequence alignment of four mitochondrial genome sequences (<italic>Cephalothrix hongkongiensis, Cephalothrix</italic>
. sp., <italic>Lineus viridis</italic>
, and <italic>Paranemertes </italic>
cf. <italic>peregrina</italic>
) retrieved from Genbank. For both species, the partial regions <italic>rrnS-rrnL </italic>
and <italic>rrnL</italic>
-<italic>cob </italic>
were amplified first. For <italic>N</italic>
. cf. <italic>mirabilis</italic>
, partial fragments of <italic>cox1 </italic>
and <italic>cox3 </italic>
genes were amplified using universal PCR primers LCO-2198/HCO-1490, cox3F/cox3R ([<xref ref-type="bibr" rid="B59">59</xref>
]; [<xref ref-type="bibr" rid="B9">9</xref>
]). These sequences were used to design specific primers to amplify the remaining mitochondrial genome fragments (<italic>cob-cox3, cox3-cox1 </italic>
and <italic>cox1-rrnS</italic>
). For <italic>Z. rubens</italic>
, the fragment of <italic>cox1-cox2 </italic>
was amplified using the universal primer LCO-2198 [<xref ref-type="bibr" rid="B59">59</xref>
] combined with the specific primer NCOX2R. Based on sequences obtained, specific primers were designed to amplify the regions <italic>cox2-rrnS, cob-cox3 </italic>
and <italic>cox3-cox1</italic>
. Conventional PCR and long PCR, cloning, and sequencing were performed as described in Chen et al. [<xref ref-type="bibr" rid="B6">6</xref>
,<xref ref-type="bibr" rid="B8">8</xref>
].</p>
<table-wrap id="T5" position="float"><label>Table 5</label>
<caption><p>PCR primers used to amplify the mitochondrial genomes of <italic>Nectonemertes </italic>
cf. <italic>mirabilis</italic>
</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th align="left">Primer name</th>
<th align="left">Sequence (5' → 3')</th>
<th align="left">References</th>
</tr>
</thead>
<tbody><tr><td align="left">Universal</td>
<td></td>
<td></td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>rrnS-rrnL</italic>
</td>
<td></td>
<td></td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left">N12SF</td>
<td align="left">TGTGCCAGCTTCCGCGGTTATAC</td>
<td align="left">Present study</td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left">N16SR</td>
<td align="left">ACGCTGTTATCCCTATGGTA</td>
<td align="left">Present study</td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>rrnL-cob</italic>
</td>
<td></td>
<td></td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left">16SarL</td>
<td align="left">CGCCTGTTTATCAAAAACAT</td>
<td align="left">[<xref ref-type="bibr" rid="B57">57</xref>
]</td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left">CytbR</td>
<td align="left">GCRTAWGCRAAWARRARTAYCAYTCWGG</td>
<td align="left">[<xref ref-type="bibr" rid="B58">58</xref>
]</td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>Nectonemertes </italic>
cf. <italic>mirabilis</italic>
</td>
<td></td>
<td></td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>cox1</italic>
</td>
<td></td>
<td></td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left">LCO-1490</td>
<td align="left">GGTCAACAAATCATAAAGATATTGG</td>
<td align="left">[<xref ref-type="bibr" rid="B59">59</xref>
]</td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left">HCO-2198</td>
<td align="left">TAAACTTCAGGGTGACCAAAAAATCA</td>
<td align="left">[<xref ref-type="bibr" rid="B59">59</xref>
]</td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>cox3</italic>
</td>
<td></td>
<td></td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left">cox3F</td>
<td align="left">TGCGWTGAGGWATAATTTTATTTATT</td>
<td align="left">[<xref ref-type="bibr" rid="B8">8</xref>
]</td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left">cox3R</td>
<td align="left">ACCAAGCAGCTGCTTCAAAACCAAA</td>
<td align="left">[<xref ref-type="bibr" rid="B8">8</xref>
]</td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>cob-cox3</italic>
</td>
<td></td>
<td></td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left">Nm cobF</td>
<td align="left">TCGGTGGATAATGCTACTTTG</td>
<td align="left">Present study</td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left">Nm COX3R</td>
<td align="left">ACCAGAAGCCAACAATACAGC</td>
<td align="left">Present study</td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>cox3-cox1</italic>
</td>
<td></td>
<td></td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left">Nm COX3F</td>
<td align="left">TGTTGGCTTCTGGTGTTAGTG</td>
<td align="left">Present study</td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left">Nm COX1R</td>
<td align="left">GAGCCTCTTTCAACAACAGCA</td>
<td align="left">Present study</td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>cox1-rrnS</italic>
</td>
<td></td>
<td></td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left">NmCOX1F</td>
<td align="left">AATCTGGTCTGGGTTGGTTGGCACTGCGTTA</td>
<td align="left">Present study</td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left">Nm12SR</td>
<td align="left">GACTCCCCTGAAAGGACATAAAACACCG</td>
<td align="left">Present study</td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>Zygeupolia rubens</italic>
</td>
<td></td>
<td></td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>cob-cox3</italic>
</td>
<td></td>
<td></td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left">Zrcob F</td>
<td align="left">CTTTGGGTTTGTTGCTGTTG</td>
<td align="left">Present study</td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left">ZrCOX3R</td>
<td align="left">GTTGAACCATAAATCCCATC</td>
<td align="left">Present study</td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>cox3-cox1</italic>
</td>
<td></td>
<td></td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left">cox3F</td>
<td align="left">TGCGWTGAGGWATAATTTTATTTATT</td>
<td align="left">[<xref ref-type="bibr" rid="B8">8</xref>
]</td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left">ZrCOX1R</td>
<td align="left">GAGCCTCTTTCAACAACAGCA</td>
<td align="left">Present study</td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>cox1-cox2</italic>
</td>
<td></td>
<td></td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left">LCO-1490</td>
<td align="left">GGTCAACAAATCATAAAGATATTGG</td>
<td align="left">[<xref ref-type="bibr" rid="B59">59</xref>
]</td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left">NCOX2R</td>
<td align="left">AAAGAATGATTWGCWCCAC</td>
<td align="left">Present study</td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left"><italic>cox2-rrnS</italic>
</td>
<td></td>
<td></td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left">ZrCOX2F</td>
<td align="left">TTTGGCTTTACCTTCTTTGC</td>
<td align="left">Present study</td>
</tr>
<tr><td colspan="3"><hr></hr>
</td>
</tr>
<tr><td align="left">Zr12SR</td>
<td align="left">AAATAAGACACCGCCAAGT</td>
<td align="left">Present study</td>
</tr>
</tbody>
</table>
</table-wrap>
</sec>
<sec><title>Sequence assemblage and annotation</title>
<p>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 <italic>Cephalothrix hongkongiensis </italic>
(GenBank #<ext-link ext-link-type="gen" xlink:href="NC_012821">NC_012821</ext-link>
), <italic>C. rufifrons </italic>
(<ext-link ext-link-type="gen" xlink:href="EF140788">EF140788</ext-link>
), <italic>Cephalothrix </italic>
sp. (<ext-link ext-link-type="gen" xlink:href="NC_014869">NC_014869</ext-link>
), <italic>Lineus viridis </italic>
(<ext-link ext-link-type="gen" xlink:href="NC_012889">NC_012889</ext-link>
), and <italic>Paranemertes </italic>
cf. <italic>peregrina </italic>
(<ext-link ext-link-type="gen" xlink:href="NC_014865">NC_014865</ext-link>
). Most tRNAs were identified by using invertebrate mitochondrial codon predictors with tRNAscan-SE 1.21 [<xref ref-type="bibr" rid="B60">60</xref>
]. 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 [<xref ref-type="bibr" rid="B61">61</xref>
], and the mitochondrial genome was visualized using CGView [<xref ref-type="bibr" rid="B62">62</xref>
].</p>
</sec>
<sec><title>Genomics analysis</title>
<p>Nucleotide composition and Relative Synonymous Codon Usage (RSCU) values were analyzed with MEGA 4.0 [<xref ref-type="bibr" rid="B63">63</xref>
]. AT- and GC-skew were determined by using the formulation of [<xref ref-type="bibr" rid="B26">26</xref>
].</p>
</sec>
<sec><title>Gene order comparisons</title>
<p>Gene orders were compared between all available nemerteans (see above), the putative bilaterian ground pattern [<xref ref-type="bibr" rid="B39">39</xref>
], <italic>Terebratulina retusa </italic>
[<xref ref-type="bibr" rid="B40">40</xref>
], <italic>Katharina tunicata </italic>
[<xref ref-type="bibr" rid="B14">14</xref>
], <italic>Phoronis psammophila </italic>
[<xref ref-type="bibr" rid="B41">41</xref>
], <italic>Perionyx excavatus </italic>
[<xref ref-type="bibr" rid="B42">42</xref>
], <italic>Urechis caupo </italic>
[<xref ref-type="bibr" rid="B43">43</xref>
] and <italic>Sipunculus nudus </italic>
[<xref ref-type="bibr" rid="B44">44</xref>
].</p>
<p>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.</p>
<p>The differences between gene orders were analysed using common intervals [<xref ref-type="bibr" rid="B38">38</xref>
], breakpoints [<xref ref-type="bibr" rid="B64">64</xref>
] and reversal distances [<xref ref-type="bibr" rid="B65">65</xref>
] implemented in the CREx tool [<xref ref-type="bibr" rid="B38">38</xref>
].</p>
</sec>
<sec><title>Phylogenetic analysis</title>
<p>The currently available near-complete and complete mitochondrial nemertean genome data (<italic>Cephalothrix </italic>
sp., <italic>C. hongkongiensis, L. viridis</italic>
, and <italic>P</italic>
. cf. <italic>peregrina</italic>
, but not the partial genome sequence of <italic>C. rufifrons</italic>
) were combined with sequences from this study for phylogenomic analyses. The nucleic acids for all 12 protein-coding genes (except <italic>atp8</italic>
, which is shortest and least conserved between the taxa) were aligned using Clustal X [<xref ref-type="bibr" rid="B66">66</xref>
] with the default settings. Ambiguously aligned portions of both alignments were excluded using Gblocks version 0.91b [<xref ref-type="bibr" rid="B67">67</xref>
] 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.</p>
<p>Based on previous studies of metazoan relationships (e.g., [<xref ref-type="bibr" rid="B68">68</xref>
-<xref ref-type="bibr" rid="B73">73</xref>
]), the following six species were selected as outgroups: a mollusc (<italic>Katharina tunicata</italic>
), a brachiopod (<italic>Terebratalia retusa</italic>
), a phoronid (<italic>Phoronis psammophila</italic>
), and three annelids (<italic>Perionyx excavatus, Sipunculus nudus </italic>
and <italic>Urechis caupo</italic>
).</p>
<p>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 [<xref ref-type="bibr" rid="B74">74</xref>
,<xref ref-type="bibr" rid="B75">75</xref>
] available on the CIPRES web portal [<xref ref-type="bibr" rid="B76">76</xref>
] with the GTRGAMMA substitution model. Support was estimated by performing 1000 bootstrap replicates. BI analysis was performed with MrBayes version 3.0b4 [<xref ref-type="bibr" rid="B77">77</xref>
,<xref ref-type="bibr" rid="B78">78</xref>
], using GTR + I + G model, a best-fit model selected by MrModeltest v2.2 [<xref ref-type="bibr" rid="B79">79</xref>
] 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.</p>
<p>Amino acid sequences were aligned with both Clustal X [<xref ref-type="bibr" rid="B66">66</xref>
] and MAFFT using the G-INS-i strategy [<xref ref-type="bibr" rid="B80">80</xref>
]. BI analyses were performed with MrBayes version 3.0b4 [<xref ref-type="bibr" rid="B77">77</xref>
,<xref ref-type="bibr" rid="B78">78</xref>
] with the mtRev + I + G model, selected by Protest 10.2 [<xref ref-type="bibr" rid="B81">81</xref>
] as optimal. We also implemented the CAT + GTR model in PhyloBayes 3 [<xref ref-type="bibr" rid="B82">82</xref>
]. The ML analysis was carried out with RAxML 7.2.7 [<xref ref-type="bibr" rid="B74">74</xref>
,<xref ref-type="bibr" rid="B75">75</xref>
] with CAT model.</p>
<p>The mitochondrial genome sequences of <italic>N</italic>
. cf. <italic>mirabilis </italic>
and <italic>Z. rubens </italic>
are deposited in GenBank under the accession numbers <ext-link ext-link-type="gen" xlink:href="HQ997772">HQ997772</ext-link>
and <ext-link ext-link-type="gen" xlink:href="HQ997773">HQ997773</ext-link>
.</p>
</sec>
</sec>
<sec><title>Abbreviations</title>
<p><italic>atp6 </italic>
and <italic>atp8</italic>
: ATP synthase subunits 6 and 8; <italic>cob</italic>
: cytochrome <italic>b</italic>
; <italic>cox1</italic>
-<italic>3</italic>
: subunits I-III of cytochrome <italic>c </italic>
oxidase; <italic>nad1</italic>
-<italic>6 </italic>
and <italic>nad4L</italic>
: NADH dehydrogenase subunits 1-6 and 4 L; <italic>rrnL </italic>
and <italic>rrnS</italic>
: the large and small subunits of ribosomal RNA; <italic>trnX</italic>
: 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.</p>
</sec>
<sec><title>Competing interests</title>
<p>The authors declare that they have no competing interests.</p>
</sec>
<sec><title>Authors' contributions</title>
<p>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. All authors read and approved the final manuscript.</p>
</sec>
<sec sec-type="supplementary-material"><title>Supplementary Material</title>
<supplementary-material content-type="local-data" id="S1"><caption><title>Additional file 1</title>
<p><bold>Figure S1</bold>
. Mitochondrial gene order (protein-coding genes and rRNAs only) of Nemertea and selected lophotrochozoan species and the putative bilaterian ground pattern (according to [<xref ref-type="bibr" rid="B39">39</xref>
]). 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. The adjacencies <italic>nad6/cob </italic>
and <italic>atp8/atp6 </italic>
are underlined. The translocation of <italic>nad2 </italic>
in the heteronemerteans and hoplonemerteans is highlight by *. Gene orders according to the following references: <italic>Cephalothrix </italic>
[<xref ref-type="bibr" rid="B6">6</xref>
,<xref ref-type="bibr" rid="B8">8</xref>
], <italic>Lineus </italic>
[<xref ref-type="bibr" rid="B7">7</xref>
], <italic>Paranemertes </italic>
[<xref ref-type="bibr" rid="B8">8</xref>
], <italic>Terebratulina </italic>
[<xref ref-type="bibr" rid="B40">40</xref>
], <italic>Katharina </italic>
[<xref ref-type="bibr" rid="B14">14</xref>
], <italic>Phoronis </italic>
[<xref ref-type="bibr" rid="B41">41</xref>
], <italic>Perionyx </italic>
[<xref ref-type="bibr" rid="B42">42</xref>
], <italic>Urechis </italic>
[<xref ref-type="bibr" rid="B43">43</xref>
], <italic>Sipunculus </italic>
[<xref ref-type="bibr" rid="B44">44</xref>
].</p>
</caption>
<media xlink:href="1471-2164-13-139-S1.DOC" mimetype="application" mime-subtype="msword"><caption><p>Click here for file</p>
</caption>
</media>
</supplementary-material>
<supplementary-material content-type="local-data" id="S2"><caption><title>Additional file 2</title>
<p><bold>Table S1</bold>
. Pairwise breakpoint distance matrix of mitochondrial gene orders of nemerteans, the bilaterian ground pattern and six other lophotrochozoans*</p>
</caption>
<media xlink:href="1471-2164-13-139-S2.DOC" mimetype="application" mime-subtype="msword"><caption><p>Click here for file</p>
</caption>
</media>
</supplementary-material>
<supplementary-material content-type="local-data" id="S3"><caption><title>Additional file 3</title>
<p><bold>Table S2</bold>
. Pairwise reversal distance matrix of mitochondrial gene orders of nemerteans, the bilaterian ground pattern and six other lophotrochozoans*</p>
</caption>
<media xlink:href="1471-2164-13-139-S3.DOC" mimetype="application" mime-subtype="msword"><caption><p>Click here for file</p>
</caption>
</media>
</supplementary-material>
</sec>
</body>
<back><sec><title>Acknowledgements</title>
<p>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 <italic>Nectonemertes </italic>
cf. <italic>mirabilis</italic>
.</p>
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