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136 lines
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<document id="F2B9C0FC6FE88CA0DC0F7686D137C05B" ID-DOI="10.1016/j.phytochem.2019.112082" ID-ISSN="1873-3700" ID-Zenodo-Dep="10483481" IM.bibliography_approvedBy="valdenar" IM.illustrations_approvedBy="valdenar" IM.materialsCitations_approvedBy="felipe" IM.metadata_approvedBy="felipe" IM.taxonomicNames_approvedBy="valdenar" IM.treatments_approvedBy="valdenar" checkinTime="1704935986447" checkinUser="felipe" docAuthor="Papaefthimiou, Dimitra, Diretto, Gianfranco, Demurtas, Olivia Costantina, Mini, Paola, Ferrante, Paola, Giuliano, Giovanni & Kanellis, Angelos K." docDate="2019" docId="713F878BFF91FFAA4768F949FEA6FE44" docLanguage="en" docName="Phytochemistry.167.112082.pdf" docOrigin="Phytochemistry 167" docSource="http://dx.doi.org/10.1016/j.phytochem.2019.112082" docStyle="DocumentStyle:9E596C34F4E94307D29315B03ACE1007.6:Phytochemistry.2014-2019.journal_article" docStyleId="9E596C34F4E94307D29315B03ACE1007" docStyleName="Phytochemistry.2014-2019.journal_article" docStyleVersion="6" docTitle="Chlamydomonas chloroplast" docType="treatment" docVersion="5" lastPageNumber="4" masterDocId="8D06FFF3FF90FFA9445AFFD7FFCCFFE9" masterDocTitle="Heterologous production of labdane-type diterpenes in the green alga Chlamydomonas reinhardtii" masterLastPageNumber="8" masterPageNumber="1" pageNumber="2" updateTime="1706544663474" updateUser="ExternalLinkService">
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<mods:title id="91F257BD5972D4C447FCD0E83921F338">Heterologous production of labdane-type diterpenes in the green alga Chlamydomonas reinhardtii</mods:title>
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<mods:namePart id="56A66C70A1A525049683DEBBDEAF92FA">Papaefthimiou, Dimitra</mods:namePart>
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<mods:affiliation id="FF5526C0D6CA57C1CE52B00206A5D0E1">∗∗ & Group of Biotechnology of Pharmaceutical Plants, Laboratory of Pharmacognosy, Department of Pharmaceutical Sciences, Aristotle University of Thessaloniki,</mods:affiliation>
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<mods:namePart id="A6FE8E9390129E764B87284B0B591987">Diretto, Gianfranco</mods:namePart>
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<mods:namePart id="A58F649042BEF038D3A40B67F3F23B4E">Demurtas, Olivia Costantina</mods:namePart>
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<mods:namePart id="42A55C9B1DD1CB923547033350104A34">Mini, Paola</mods:namePart>
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<mods:namePart id="1A713EF180635F59DF646E62D7A9118D">Ferrante, Paola</mods:namePart>
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<mods:namePart id="904387BE3815FFEC74BD66D093D144EB">Giuliano, Giovanni</mods:namePart>
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<mods:namePart id="E31328B20EA350837CA5D9A5993F9B0E">Kanellis, Angelos K.</mods:namePart>
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<treatment id="713F878BFF91FFAA4768F949FEA6FE44" ID-DOI="http://doi.org/10.5281/zenodo.10581204" ID-Zenodo-Dep="10581204" LSID="urn:lsid:plazi:treatment:713F878BFF91FFAA4768F949FEA6FE44" httpUri="http://treatment.plazi.org/id/713F878BFF91FFAA4768F949FEA6FE44" lastPageId="3" lastPageNumber="4" pageId="1" pageNumber="2">
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<subSubSection id="B18C6516FF91FFA84768F949FBA0F925" pageId="1" pageNumber="2" type="nomenclature">
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<paragraph id="F929369DFF91FFA84768F949FBA0F925" blockId="1.[818,1449,1694,1713]" lastBlockId="1.[818,1132,1721,1740]" pageId="1" pageNumber="2">
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<heading id="A26181F1FF91FFA84768F949FC4BF925" bold="true" fontSize="36" level="1" pageId="1" pageNumber="2" reason="1">
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2.1. Transformation of the
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<taxonomicName id="3E964D1EFF91FFA84071F949FB72F958" ID-CoL="3NGJ" authority="Ehrenberg, 1833" box="[1067,1214,1694,1713]" class="Chlorophyceae" family="Chlamydomonadaceae" genus="Chlamydomonas" kingdom="Plantae" order="Chlamydomonadales" pageId="1" pageNumber="2" phylum="Chlorophyta" rank="genus">Chlamydomonas</taxonomicName>
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chloroplast with a codonoptimized
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</heading>
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CcCLS gene (CO-CcCLS)
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</emphasis>
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</paragraph>
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<subSubSection id="B18C6516FF91FFAA4709F926FEA6FE44" lastPageId="3" lastPageNumber="4" pageId="1" pageNumber="2" type="description">
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<paragraph id="F929369DFF91FFA84709F926FA03F821" blockId="1.[818,1487,1777,1992]" pageId="1" pageNumber="2">
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The CDS of the
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<taxonomicName id="3E964D1EFF91FFA847A2F926FB51F8ED" authority="CcCLS" authorityName="CcCLS" box="[1016,1181,1777,1796]" class="Magnoliopsida" family="Cistaceae" genus="Cistus" kingdom="Plantae" order="Malvales" pageId="1" pageNumber="2" phylum="Tracheophyta" rank="species" species="creticus">
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<emphasis id="CBE2EA8FFF91FFA847A2F926FB51F8ED" bold="true" box="[1016,1181,1777,1796]" italics="true" pageId="1" pageNumber="2">C. creticus CcCLS</emphasis>
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</taxonomicName>
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gene was codon-optimized for improved expression in the
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<taxonomicName id="3E964D1EFF91FFA84001F8DAFB17F8C9" box="[1115,1243,1805,1824]" class="Chlorophyceae" family="Chlamydomonadaceae" genus="Chlamydomonas" kingdom="Plantae" order="Chlamydomonadales" pageId="1" pageNumber="2" phylum="Chlorophyta" rank="species" species="reinhardtii">
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<emphasis id="CBE2EA8FFF91FFA84001F8DAFB17F8C9" bold="true" box="[1115,1243,1805,1824]" italics="true" pageId="1" pageNumber="2">C. reinhardtii</emphasis>
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</taxonomicName>
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chloroplast (
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<emphasis id="CBE2EA8FFF91FFA84130F8DAFA0BF8C9" bold="true" box="[1386,1479,1805,1824]" italics="true" pageId="1" pageNumber="2">CO-CcCLS</emphasis>
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) (Supplementary
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<figureCitation id="61AD2A18FF91FFA8478EF8FEFBDCF8D5" box="[980,1040,1833,1852]" captionStart="Fig" captionStartId="1.[100,130,964,981]" captionTargetBox="[264,1323,153,940]" captionTargetId="figure-686@1.[263,1325,152,941]" captionTargetPageId="1" captionText="Fig. 1. Proposed pathway of labdane-type diterpenes predominant in Cistus creticus L. resin. A protonation-initiated cyclization, catalyzed by CcCLS, converts GGDP to the stable bicyclic intermediate copal-8-ol diphosphate. The formation of labda-13-ene-8α,15-diol, ent-manoyl oxide, ent-13-epi manoyl oxide and sclareol are probably due to the activity of non-specific terpenoid phosphatases. Abbreviations: CcCLS, Cistus creticus copal-8-ol diphosphate synthase." figureDoi="http://doi.org/10.5281/zenodo.10483483" httpUri="https://zenodo.org/record/10483483/files/figure.png" pageId="1" pageNumber="2">Fig. 1</figureCitation>
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).
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<emphasis id="CBE2EA8FFF91FFA84071F8FEFB44F8D5" bold="true" box="[1067,1160,1833,1852]" italics="true" pageId="1" pageNumber="2">CO-CcCLS</emphasis>
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was cloned without the transit peptide as an N-terminal fusion with the FLAG–tag in the pCG2-FLAG chloroplast expression vector, under the control of the
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<emphasis id="CBE2EA8FFF91FFA8416FF8B6FAABF89D" bold="true" box="[1333,1383,1889,1908]" italics="true" pageId="1" pageNumber="2">PSBD</emphasis>
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promoter/ 5′UTR and
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<emphasis id="CBE2EA8FFF91FFA847FAF8AAFC1EF879" bold="true" box="[928,978,1917,1936]" italics="true" pageId="1" pageNumber="2">PSBA</emphasis>
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terminator/3′UTR (
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<figureCitation id="61AD2A18FF91FFA840CFF8AAFB13F879" box="[1173,1247,1917,1936]" captionStart="Fig" captionStartId="2.[100,130,1648,1665]" captionTargetBox="[264,1324,153,1624]" captionTargetId="figure-0@2.[263,1325,152,1625]" captionTargetPageId="2" captionText="Fig. 2. Chloroplast transformation of C. reinhardtii with recombinant CO-CcCLS gene, and verification of transgene integration and homoplasmy. A. Schematic map of pCG2-FLAG-CO-CcCLS vector used for chloroplast transformation. The length of each genetic element is indicated in each box. atpA prom.: Promoter of the α- subunit of the chloroplast ATP synthase; aadA: CDS of spectinomycin resistance gene; rbcL Ter.: ribulose bisphosphate carboxylase large chain Terminator; psbA Ter.: Photosystem II protein D1 Terminator; CO-CcCLS: Codon adapted CLS gene; FLAG: Flag Tag; psbD prom.: Photosystem II protein D2 promoter. B. Integration scheme of pCG2-FLAG-CO-CcCLS plasmid in the chloroplast genome. Primers Chla1-for/Chla1-rev (amplicon 1: length 1358 bp) and Chla2-for/Chla2-rev (amplicon2: length 2519 bp) were used to test correct integration in the chloroplast genome while primers Chla3-for/Chla3-rev were used to check homoplasmy (amplicon 3: length 954 bp). The absence of amplicon 3 indicates that the transformants are homoplasmic for the presence of transgene. C. PCR results of seventeen representative transformants. Transformants indicated in red have correct plasmid integration and are homoplasmic. M: 1 kb DNA ladder (NEB, catalog number N3232S)." figureDoi="http://doi.org/10.5281/zenodo.10483485" httpUri="https://zenodo.org/record/10483485/files/figure.png" pageId="1" pageNumber="2">Fig. 2A</figureCitation>
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). The obtained construct (pCG2-FLAG-
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<emphasis id="CBE2EA8FFF91FFA847F5F84EFBC0F845" bold="true" box="[943,1036,1945,1964]" italics="true" pageId="1" pageNumber="2">CO-CcCLS</emphasis>
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) was introduced in the chloroplast of the cell wall-less cw15 strain using glass beads transformation (Demurtas et al.,
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</paragraph>
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<caption id="ADE96615FF92FFAB443EF9A7FABCF8DA" ID-DOI="http://doi.org/10.5281/zenodo.10483485" ID-Zenodo-Dep="10483485" httpUri="https://zenodo.org/record/10483485/files/figure.png" pageId="2" pageNumber="3" startId="2.[100,130,1648,1665]" targetBox="[264,1324,153,1624]" targetPageId="2" targetType="figure">
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<paragraph id="F929369DFF92FFAB443EF9A7FABCF8DA" blockId="2.[100,1488,1647,1843]" pageId="2" pageNumber="3">
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<emphasis id="CBE2EA8FFF92FFAB443EF9A7FF51F968" bold="true" box="[100,157,1648,1665]" pageId="2" pageNumber="3">Fig. 2.</emphasis>
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Chloroplast transformation of
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<taxonomicName id="3E964D1EFF92FFAB45F8F9A7FDDFF968" box="[418,531,1648,1665]" class="Chlorophyceae" family="Chlamydomonadaceae" genus="Chlamydomonas" kingdom="Plantae" order="Chlamydomonadales" pageId="2" pageNumber="3" phylum="Chlorophyta" rank="species" species="reinhardtii">C. reinhardtii</taxonomicName>
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with recombinant CO-CcCLS gene, and verification of transgene integration and homoplasmy. A. Schematic map of pCG2-FLAG-CO-CcCLS vector used for chloroplast transformation. The length of each genetic element is indicated in each box. atpA prom.: Promoter of the α- subunit of the
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<taxonomicName id="3E964D1EFF92FFAB4487F974FEAFF95D" authority="ATP" authorityName="ATP" box="[221,355,1699,1716]" class="Chlorophyceae" family="Chlamydomonadaceae" genus="Chlamydomonas" kingdom="Plantae" order="Chlamydomonadales" pageId="2" pageNumber="3" phylum="Chlorophyta" rank="species" species="chloroplast">chloroplast ATP</taxonomicName>
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synthase; aadA: CDS of spectinomycin resistance gene; rbcL Ter.: ribulose bisphosphate carboxylase large chain Terminator; psbA Ter.: Photosystem II protein D1 Terminator; CO-CcCLS: Codon adapted CLS gene; FLAG: Flag Tag; psbD prom.: Photosystem II protein D2 promoter. B. Integration scheme of pCG2-FLAG-CO-CcCLS plasmid in the chloroplast genome. Primers Chla1-for/Chla1-rev (amplicon 1: length 1358 bp) and Chla2-for/Chla2-rev (amplicon2: length 2519 bp) were used to test correct integration in the chloroplast genome while primers Chla3-for/Chla3-rev were used to check homoplasmy (amplicon 3: length 954 bp). The absence of amplicon 3 indicates that the transformants are homoplasmic for the presence of transgene. C. PCR results of seventeen representative transformants. Transformants indicated in red have correct plasmid integration and are homoplasmic. M: 1 kb DNA ladder (NEB, catalog number N3232S).
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</paragraph>
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</caption>
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<paragraph id="F929369DFF93FFAA443EFF48FEA6FE44" blockId="3.[100,770,159,429]" pageId="3" pageNumber="4">
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2013). Chloroplast transformants were selected on TAP agar plates supplemented with 100 μg/ml spectinomycin and subcultured for 10 rounds in the same medium to eliminate all wild-type copies of the chloroplast genome, a condition known as homoplasmy. Correct integration in the chloroplast genome and homoplasmy were verified by PCR with appropriate primers (
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<figureCitation id="61AD2A18FF93FFAA45D7FEFCFE0EFED7" box="[397,450,299,318]" captionStart="Fig" captionStartId="2.[100,130,1648,1665]" captionTargetBox="[264,1324,153,1624]" captionTargetId="figure-0@2.[263,1325,152,1625]" captionTargetPageId="2" captionText="Fig. 2. Chloroplast transformation of C. reinhardtii with recombinant CO-CcCLS gene, and verification of transgene integration and homoplasmy. A. Schematic map of pCG2-FLAG-CO-CcCLS vector used for chloroplast transformation. The length of each genetic element is indicated in each box. atpA prom.: Promoter of the α- subunit of the chloroplast ATP synthase; aadA: CDS of spectinomycin resistance gene; rbcL Ter.: ribulose bisphosphate carboxylase large chain Terminator; psbA Ter.: Photosystem II protein D1 Terminator; CO-CcCLS: Codon adapted CLS gene; FLAG: Flag Tag; psbD prom.: Photosystem II protein D2 promoter. B. Integration scheme of pCG2-FLAG-CO-CcCLS plasmid in the chloroplast genome. Primers Chla1-for/Chla1-rev (amplicon 1: length 1358 bp) and Chla2-for/Chla2-rev (amplicon2: length 2519 bp) were used to test correct integration in the chloroplast genome while primers Chla3-for/Chla3-rev were used to check homoplasmy (amplicon 3: length 954 bp). The absence of amplicon 3 indicates that the transformants are homoplasmic for the presence of transgene. C. PCR results of seventeen representative transformants. Transformants indicated in red have correct plasmid integration and are homoplasmic. M: 1 kb DNA ladder (NEB, catalog number N3232S)." figureDoi="http://doi.org/10.5281/zenodo.10483485" httpUri="https://zenodo.org/record/10483485/files/figure.png" pageId="3" pageNumber="4">Fig. 2</figureCitation>
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) (
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<bibRefCitation id="9D074B6CFF93FFAA4582FEFCFD6FFED7" author="Demurtas, O. C. & Massa, S. & Ferrante, P. & Venuti, A. & Franconi, R. & Giuliano, G." box="[472,675,299,318]" pageId="3" pageNumber="4" pagination="61473" refId="ref6525" refString="Demurtas, O. C., Massa, S., Ferrante, P., Venuti, A., Franconi, R., Giuliano, G., 2013. A Chlamydomonas - derived human papillomavirus 16 E 7 vaccine induces specific tumor protection. PLoS One 8, e 61473. https: // doi. org / 10.1371 / journal. pone. 0061473." type="journal article" year="2013">Demurtas et al., 2013</bibRefCitation>
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). In total, 79 transformants were tested by PCR and 49 of them showed correct integration and homoplasmy.
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<figureCitation id="61AD2A18FF93FFAA45DFFEB4FE04FE9F" box="[389,456,355,374]" captionStart="Fig" captionStartId="2.[100,130,1648,1665]" captionTargetBox="[264,1324,153,1624]" captionTargetId="figure-0@2.[263,1325,152,1625]" captionTargetPageId="2" captionText="Fig. 2. Chloroplast transformation of C. reinhardtii with recombinant CO-CcCLS gene, and verification of transgene integration and homoplasmy. A. Schematic map of pCG2-FLAG-CO-CcCLS vector used for chloroplast transformation. The length of each genetic element is indicated in each box. atpA prom.: Promoter of the α- subunit of the chloroplast ATP synthase; aadA: CDS of spectinomycin resistance gene; rbcL Ter.: ribulose bisphosphate carboxylase large chain Terminator; psbA Ter.: Photosystem II protein D1 Terminator; CO-CcCLS: Codon adapted CLS gene; FLAG: Flag Tag; psbD prom.: Photosystem II protein D2 promoter. B. Integration scheme of pCG2-FLAG-CO-CcCLS plasmid in the chloroplast genome. Primers Chla1-for/Chla1-rev (amplicon 1: length 1358 bp) and Chla2-for/Chla2-rev (amplicon2: length 2519 bp) were used to test correct integration in the chloroplast genome while primers Chla3-for/Chla3-rev were used to check homoplasmy (amplicon 3: length 954 bp). The absence of amplicon 3 indicates that the transformants are homoplasmic for the presence of transgene. C. PCR results of seventeen representative transformants. Transformants indicated in red have correct plasmid integration and are homoplasmic. M: 1 kb DNA ladder (NEB, catalog number N3232S)." figureDoi="http://doi.org/10.5281/zenodo.10483485" httpUri="https://zenodo.org/record/10483485/files/figure.png" pageId="3" pageNumber="4">Fig. 2C</figureCitation>
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shows the PCR results of seventeen representative transformants, where only twelve had correct integration and homoplasmy.
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</paragraph>
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</subSubSection>
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</treatment>
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