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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Testing the performance of a fragment of the COI gene to identify western Palaearctic stag beetle species (Coleoptera, Lucanidae)</title><author><name sortKey="Cox, Karen" sort="Cox, Karen" uniqKey="Cox K" first="Karen" last="Cox">Karen Cox</name><affiliation><nlm:aff id="A1">Research Institute for Nature and Forest, Gaverstraat 4, B-9500 Geraardsbergen, Belgium</nlm:aff></affiliation></author><author><name sortKey="Thomaes, Arno" sort="Thomaes, Arno" uniqKey="Thomaes A" first="Arno" last="Thomaes">Arno Thomaes</name><affiliation><nlm:aff id="A1">Research Institute for Nature and Forest, Gaverstraat 4, B-9500 Geraardsbergen, Belgium</nlm:aff></affiliation></author><author><name sortKey="Antonini, Gloria" sort="Antonini, Gloria" uniqKey="Antonini G" first="Gloria" last="Antonini">Gloria Antonini</name><affiliation><nlm:aff id="A2">Department of Biology and Biotechnology “Charles Darwin”, Sapienza - University of Rome, via A. Borelli 50, I-00161 Rome, Italy</nlm:aff></affiliation></author><author><name sortKey="Zilioli, Michele" sort="Zilioli, Michele" uniqKey="Zilioli M" first="Michele" last="Zilioli">Michele Zilioli</name><affiliation><nlm:aff id="A3">Natural History Museum, Entomological section, Corso Venezia 55, I-20121 Milano, Italy</nlm:aff></affiliation></author><author><name sortKey="De Gelas, Koen" sort="De Gelas, Koen" uniqKey="De Gelas K" first="Koen" last="De Gelas">Koen De Gelas</name><affiliation><nlm:aff id="A1">Research Institute for Nature and Forest, Gaverstraat 4, B-9500 Geraardsbergen, Belgium</nlm:aff></affiliation><affiliation><nlm:aff id="A4">Royal Belgian Institute of Natural Sciences, Vautierstraat 29, B-1000 Brussels, Belgium</nlm:aff></affiliation></author><author><name sortKey="Harvey, Deborah" sort="Harvey, Deborah" uniqKey="Harvey D" first="Deborah" last="Harvey">Deborah Harvey</name><affiliation><nlm:aff id="A5">School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey, UK</nlm:aff></affiliation></author><author><name sortKey="Solano, Emanuela" sort="Solano, Emanuela" uniqKey="Solano E" first="Emanuela" last="Solano">Emanuela Solano</name><affiliation><nlm:aff id="A2">Department of Biology and Biotechnology “Charles Darwin”, Sapienza - University of Rome, via A. Borelli 50, I-00161 Rome, Italy</nlm:aff></affiliation></author><author><name sortKey="Audisio, Paolo" sort="Audisio, Paolo" uniqKey="Audisio P" first="Paolo" last="Audisio">Paolo Audisio</name><affiliation><nlm:aff id="A2">Department of Biology and Biotechnology “Charles Darwin”, Sapienza - University of Rome, via A. Borelli 50, I-00161 Rome, Italy</nlm:aff></affiliation></author><author><name sortKey="Mckeown, Niall" sort="Mckeown, Niall" uniqKey="Mckeown N" first="Niall" last="Mckeown">Niall Mckeown</name><affiliation><nlm:aff id="A6">Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University, Penglais, Aberystwyth, UK</nlm:aff></affiliation></author><author><name sortKey="Shaw, Paul" sort="Shaw, Paul" uniqKey="Shaw P" first="Paul" last="Shaw">Paul Shaw</name><affiliation><nlm:aff id="A6">Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University, Penglais, Aberystwyth, UK</nlm:aff></affiliation></author><author><name sortKey="Minetti, Robert" sort="Minetti, Robert" uniqKey="Minetti R" first="Robert" last="Minetti">Robert Minetti</name><affiliation><nlm:aff id="A7">7 Avenue Marc Sangnier, 13600 La Ciotat, France</nlm:aff></affiliation></author><author><name sortKey="Bartolozzi, Luca" sort="Bartolozzi, Luca" uniqKey="Bartolozzi L" first="Luca" last="Bartolozzi">Luca Bartolozzi</name><affiliation><nlm:aff id="A8">Natural History Museum, Zoological Section “La Specola”, via Romana 17, 50125 Firenze, Italy</nlm:aff></affiliation></author><author><name sortKey="Mergeay, Joachim" sort="Mergeay, Joachim" uniqKey="Mergeay J" first="Joachim" last="Mergeay">Joachim Mergeay</name><affiliation><nlm:aff id="A1">Research Institute for Nature and Forest, Gaverstraat 4, B-9500 Geraardsbergen, Belgium</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">24453554</idno><idno type="pmc">3890674</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3890674</idno><idno type="RBID">PMC:3890674</idno><idno type="doi">10.3897/zookeys.365.5526</idno><date when="2013">2013</date><idno type="wicri:Area/Pmc/Corpus">000272</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000272</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Testing the performance of a fragment of the COI gene to identify western Palaearctic stag beetle species (Coleoptera, Lucanidae)</title><author><name sortKey="Cox, Karen" sort="Cox, Karen" uniqKey="Cox K" first="Karen" last="Cox">Karen Cox</name><affiliation><nlm:aff id="A1">Research Institute for Nature and Forest, Gaverstraat 4, B-9500 Geraardsbergen, Belgium</nlm:aff></affiliation></author><author><name sortKey="Thomaes, Arno" sort="Thomaes, Arno" uniqKey="Thomaes A" first="Arno" last="Thomaes">Arno Thomaes</name><affiliation><nlm:aff id="A1">Research Institute for Nature and Forest, Gaverstraat 4, B-9500 Geraardsbergen, Belgium</nlm:aff></affiliation></author><author><name sortKey="Antonini, Gloria" sort="Antonini, Gloria" uniqKey="Antonini G" first="Gloria" last="Antonini">Gloria Antonini</name><affiliation><nlm:aff id="A2">Department of Biology and Biotechnology “Charles Darwin”, Sapienza - University of Rome, via A. Borelli 50, I-00161 Rome, Italy</nlm:aff></affiliation></author><author><name sortKey="Zilioli, Michele" sort="Zilioli, Michele" uniqKey="Zilioli M" first="Michele" last="Zilioli">Michele Zilioli</name><affiliation><nlm:aff id="A3">Natural History Museum, Entomological section, Corso Venezia 55, I-20121 Milano, Italy</nlm:aff></affiliation></author><author><name sortKey="De Gelas, Koen" sort="De Gelas, Koen" uniqKey="De Gelas K" first="Koen" last="De Gelas">Koen De Gelas</name><affiliation><nlm:aff id="A1">Research Institute for Nature and Forest, Gaverstraat 4, B-9500 Geraardsbergen, Belgium</nlm:aff></affiliation><affiliation><nlm:aff id="A4">Royal Belgian Institute of Natural Sciences, Vautierstraat 29, B-1000 Brussels, Belgium</nlm:aff></affiliation></author><author><name sortKey="Harvey, Deborah" sort="Harvey, Deborah" uniqKey="Harvey D" first="Deborah" last="Harvey">Deborah Harvey</name><affiliation><nlm:aff id="A5">School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey, UK</nlm:aff></affiliation></author><author><name sortKey="Solano, Emanuela" sort="Solano, Emanuela" uniqKey="Solano E" first="Emanuela" last="Solano">Emanuela Solano</name><affiliation><nlm:aff id="A2">Department of Biology and Biotechnology “Charles Darwin”, Sapienza - University of Rome, via A. Borelli 50, I-00161 Rome, Italy</nlm:aff></affiliation></author><author><name sortKey="Audisio, Paolo" sort="Audisio, Paolo" uniqKey="Audisio P" first="Paolo" last="Audisio">Paolo Audisio</name><affiliation><nlm:aff id="A2">Department of Biology and Biotechnology “Charles Darwin”, Sapienza - University of Rome, via A. Borelli 50, I-00161 Rome, Italy</nlm:aff></affiliation></author><author><name sortKey="Mckeown, Niall" sort="Mckeown, Niall" uniqKey="Mckeown N" first="Niall" last="Mckeown">Niall Mckeown</name><affiliation><nlm:aff id="A6">Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University, Penglais, Aberystwyth, UK</nlm:aff></affiliation></author><author><name sortKey="Shaw, Paul" sort="Shaw, Paul" uniqKey="Shaw P" first="Paul" last="Shaw">Paul Shaw</name><affiliation><nlm:aff id="A6">Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University, Penglais, Aberystwyth, UK</nlm:aff></affiliation></author><author><name sortKey="Minetti, Robert" sort="Minetti, Robert" uniqKey="Minetti R" first="Robert" last="Minetti">Robert Minetti</name><affiliation><nlm:aff id="A7">7 Avenue Marc Sangnier, 13600 La Ciotat, France</nlm:aff></affiliation></author><author><name sortKey="Bartolozzi, Luca" sort="Bartolozzi, Luca" uniqKey="Bartolozzi L" first="Luca" last="Bartolozzi">Luca Bartolozzi</name><affiliation><nlm:aff id="A8">Natural History Museum, Zoological Section “La Specola”, via Romana 17, 50125 Firenze, Italy</nlm:aff></affiliation></author><author><name sortKey="Mergeay, Joachim" sort="Mergeay, Joachim" uniqKey="Mergeay J" first="Joachim" last="Mergeay">Joachim Mergeay</name><affiliation><nlm:aff id="A1">Research Institute for Nature and Forest, Gaverstraat 4, B-9500 Geraardsbergen, Belgium</nlm:aff></affiliation></author></analytic><series><title level="j">ZooKeys</title><idno type="ISSN">1313-2989</idno><idno type="eISSN">1313-2970</idno><imprint><date when="2013">2013</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><label>Abstract</label><p>The taxonomy of stag beetles (Coleoptera: Lucanidae) remains challenging, mainly due to the sexual dimorphism and the strong allometry in males. Such conjecture confounds taxonomic based conservation efforts that are urgently needed due to numerous threats to stag beetle biodiversity. Molecular tools could help solve the problem of identification of the different recognized taxa in the “<pmc-comment>PageBreak</pmc-comment><italic>Lucanus cervus</italic> complex” and in some related Palaearctic species. We investigated the potential use of a 670 bp region at the 3’ end of the mitochondrial cytochrome <italic>c</italic> oxidase subunit I gene (COI) for barcoding purposes (different from the standard COI barcoding region). Well resolved species and subspecies were <italic>L. tetraodon</italic>, <italic>L. cervusakbesianus</italic>, <italic>L. c. laticornis</italic>, as well as the two eastern Asian outgroup taxa <italic>L. formosanus</italic> and <italic>L. hermani</italic>. Conversely, certain taxa could not be distinguished from each other based on K2P-distances and tree topologies: <italic>L. c. fabiani</italic> / <italic>L. (P.) barbarossa</italic>, <italic>L. c. judaicus</italic> / an unknown <italic>Lucanus</italic> species, <italic>L. c. cervus</italic> / <italic>L. c. turcicus</italic> / <italic>L. c. pentaphyllus</italic> / <italic>L. (P.) macrophyllus</italic> / <italic>L. ibericus</italic>. The relative roles of phenotypic plasticity, recurrent hybridisation and incomplete lineage sorting underlying taxonomic and phylogenetic discordances are discussed.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Aljanabi, Sm" uniqKey="Aljanabi S">SM Aljanabi</name></author><author><name sortKey="Martinez, I" uniqKey="Martinez I">I Martinez</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Avise, Jc" uniqKey="Avise J">JC Avise</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Baraud, J" uniqKey="Baraud J">J Baraud</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bartolozzi, L" uniqKey="Bartolozzi L">L Bartolozzi</name></author><author><name sortKey="Sprecher Uebersax, E" uniqKey="Sprecher Uebersax E">E Sprecher-Uebersax</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Benesh, B" uniqKey="Benesh 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Zookeys</journal-id><journal-id journal-id-type="iso-abbrev">Zookeys</journal-id><journal-id journal-id-type="publisher-id">ZooKeys</journal-id><journal-title-group><journal-title>ZooKeys</journal-title></journal-title-group><issn pub-type="ppub">1313-2989</issn><issn pub-type="epub">1313-2970</issn><publisher><publisher-name>Pensoft Publishers</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">24453554</article-id><article-id pub-id-type="pmc">3890674</article-id><article-id pub-id-type="doi">10.3897/zookeys.365.5526</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Testing the performance of a fragment of the COI gene to identify western Palaearctic stag beetle species (Coleoptera, Lucanidae)</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Cox</surname><given-names>Karen</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Thomaes</surname><given-names>Arno</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Antonini</surname><given-names>Gloria</given-names></name><xref ref-type="aff" rid="A2">2</xref></contrib><contrib contrib-type="author"><name><surname>Zilioli</surname><given-names>Michele</given-names></name><xref ref-type="aff" rid="A3">3</xref></contrib><contrib contrib-type="author"><name><surname>De Gelas</surname><given-names>Koen</given-names></name><xref ref-type="aff" rid="A1">1</xref><xref ref-type="aff" rid="A4">4</xref></contrib><contrib contrib-type="author"><name><surname>Harvey</surname><given-names>Deborah</given-names></name><xref ref-type="aff" rid="A5">5</xref></contrib><contrib contrib-type="author"><name><surname>Solano</surname><given-names>Emanuela</given-names></name><xref ref-type="aff" rid="A2">2</xref></contrib><contrib contrib-type="author"><name><surname>Audisio</surname><given-names>Paolo</given-names></name><xref ref-type="aff" rid="A2">2</xref></contrib><contrib contrib-type="author"><name><surname>McKeown</surname><given-names>Niall</given-names></name><xref ref-type="aff" rid="A6">6</xref></contrib><contrib contrib-type="author"><name><surname>Shaw</surname><given-names>Paul</given-names></name><xref ref-type="aff" rid="A6">6</xref></contrib><contrib contrib-type="author"><name><surname>Minetti</surname><given-names>Robert</given-names></name><xref ref-type="aff" rid="A7">7</xref></contrib><contrib contrib-type="author"><name><surname>Bartolozzi</surname><given-names>Luca</given-names></name><xref ref-type="aff" rid="A8">8</xref></contrib><contrib contrib-type="author"><name><surname>Mergeay</surname><given-names>Joachim</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib></contrib-group><aff id="A1"><label>1</label>Research Institute for Nature and Forest, Gaverstraat 4, B-9500 Geraardsbergen, Belgium</aff><aff id="A2"><label>2</label>Department of Biology and Biotechnology “Charles Darwin”, Sapienza - University of Rome, via A. Borelli 50, I-00161 Rome, Italy</aff><aff id="A3"><label>3</label>Natural History Museum, Entomological section, Corso Venezia 55, I-20121 Milano, Italy</aff><aff id="A4"><label>4</label>Royal Belgian Institute of Natural Sciences, Vautierstraat 29, B-1000 Brussels, Belgium</aff><aff id="A5"><label>5</label>School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey, UK</aff><aff id="A6"><label>6</label>Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University, Penglais, Aberystwyth, UK</aff><aff id="A7"><label>7</label>7 Avenue Marc Sangnier, 13600 La Ciotat, France</aff><aff id="A8"><label>8</label>Natural History Museum, Zoological Section “La Specola”, via Romana 17, 50125 Firenze, Italy</aff><author-notes><corresp>Corresponding author: Karen Cox (<email xlink:type="simple">karen.cox@inbo.be</email>)</corresp><fn fn-type="edited-by"><p>Academic editor: M. de Meyer</p></fn></author-notes><pub-date pub-type="collection"><year>2013</year></pub-date><pub-date pub-type="epub"><day>30</day><month>12</month><year>2013</year></pub-date><issue>365</issue><fpage>105</fpage><lpage>126</lpage><history><date date-type="received"><day>15</day><month>5</month><year>2013</year></date><date date-type="accepted"><day>16</day><month>10</month><year>2013</year></date></history><permissions><copyright-statement>Karen Cox, Arno Thomaes, Gloria Antonini, Michele Zilioli, Koen De Gelas, Deborah Harvey, Emanuela Solano, Paolo Audisio, Niall McKeown, Paul Shaw, Robert Minetti, Luca Bartolozzi, Joachim Mergeay</copyright-statement><license license-type="creative-commons-attribution" xlink:href="http://creativecommons.org/licenses/by/4.0"><license-p>This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><abstract><label>Abstract</label><p>The taxonomy of stag beetles (Coleoptera: Lucanidae) remains challenging, mainly due to the sexual dimorphism and the strong allometry in males. Such conjecture confounds taxonomic based conservation efforts that are urgently needed due to numerous threats to stag beetle biodiversity. Molecular tools could help solve the problem of identification of the different recognized taxa in the “<pmc-comment>PageBreak</pmc-comment><italic>Lucanus cervus</italic> complex” and in some related Palaearctic species. We investigated the potential use of a 670 bp region at the 3’ end of the mitochondrial cytochrome <italic>c</italic> oxidase subunit I gene (COI) for barcoding purposes (different from the standard COI barcoding region). Well resolved species and subspecies were <italic>L. tetraodon</italic>, <italic>L. cervusakbesianus</italic>, <italic>L. c. laticornis</italic>, as well as the two eastern Asian outgroup taxa <italic>L. formosanus</italic> and <italic>L. hermani</italic>. Conversely, certain taxa could not be distinguished from each other based on K2P-distances and tree topologies: <italic>L. c. fabiani</italic> / <italic>L. (P.) barbarossa</italic>, <italic>L. c. judaicus</italic> / an unknown <italic>Lucanus</italic> species, <italic>L. c. cervus</italic> / <italic>L. c. turcicus</italic> / <italic>L. c. pentaphyllus</italic> / <italic>L. (P.) macrophyllus</italic> / <italic>L. ibericus</italic>. The relative roles of phenotypic plasticity, recurrent hybridisation and incomplete lineage sorting underlying taxonomic and phylogenetic discordances are discussed.</p></abstract><kwd-group><label>Keywords</label><kwd><italic>Lucanus</italic> spp.</kwd><kwd>Stag beetle</kwd><kwd>Western Palaearctic</kwd><kwd>DNA barcoding</kwd><kwd>COI</kwd></kwd-group></article-meta></front><body><sec><title>Introduction</title><p><named-content content-type="taxon-name">Lucanidae</named-content> Latreille, 1804 is a family of <named-content content-type="taxon-name">Coleoptera</named-content> showing in most species pronounced sexual dimorphism and strong external morphological allometry in males. The species of the Holarctic and Oriental distributed genus <italic><named-content content-type="taxon-name">Lucanus</named-content></italic> Scopoli, 1763 are renowned for the striking appearance of the males. With their large body size and prominent mandibles, the male stag beetles are very popular among amateur entomologists and as terrarium pets, mainly in Japan. Currently, there are more than 90 <italic><named-content content-type="taxon-name">Lucanus</named-content></italic> species described, however, validity of these designations is considered questionable in many cases. Sexual dimorphism and size variation complicate the taxonomy (<xref ref-type="bibr" rid="B12">Didier and Séguy 1953</xref>, <xref ref-type="bibr" rid="B9">Clark 1977</xref>, <xref ref-type="bibr" rid="B24">Harvey and Gange 2006</xref>), as does the lack of informative phenotypic characters among larvae. Consequently, their classification has changed over time and is still under discussion. In this study we focus on taxa of the <italic><named-content content-type="taxon-name">Lucanus</named-content></italic> species in the western Palaearctic.</p><p>The genus <italic><named-content content-type="taxon-name">Lucanus</named-content></italic> is subdivided into the subgenera <italic><named-content content-type="taxon-name">Lucanus</named-content></italic> sensu stricto and <italic><named-content content-type="taxon-name">Pseudolucanus</named-content></italic> Hope & Westwood, 1845. Members of the latter have a peculiar stout body and substantial analogy of morphology that makes it quite easy to distinguish them from members of the subgenus <italic><named-content content-type="taxon-name">Lucanus</named-content></italic> (<xref ref-type="bibr" rid="B47">Planet 1899</xref>). The male mandibles of <italic><named-content content-type="taxon-name">Pseudolucanus</named-content></italic> are sickle shaped, their internal edge has a single denticle in most species (<italic><named-content content-type="taxon-name">Lucanus</named-content></italic> has small denticles and one large denticle) and the apex is usually simple (<italic><named-content content-type="taxon-name">Lucanus</named-content></italic> is mostly bifid) (<xref ref-type="bibr" rid="B47">Planet 1899</xref>, <xref ref-type="bibr" rid="B3">Baraud 1993</xref>). Furthermore, the integument of <italic><named-content content-type="taxon-name">Pseudolucanus</named-content></italic> is relatively smooth with scattered and superficial punctuation whereas it is more stippled in <italic><named-content content-type="taxon-name">Lucanus</named-content></italic>. Also, the sides of the pronotum of <italic><named-content content-type="taxon-name">Pseudolucanus</named-content></italic> are strongly sinuate before the posterior angles (<xref ref-type="bibr" rid="B3">Baraud 1993</xref>). Previous studies (<xref ref-type="bibr" rid="B12">Didier and Séguy 1953</xref>, <xref ref-type="bibr" rid="B5">Benesh 1960</xref>, <xref ref-type="bibr" rid="B36">Krajcik 2001</xref>, <xref ref-type="bibr" rid="B4">Bartolozzi and Sprecher-Uebersax 2006</xref>, <xref ref-type="bibr" rid="B23">Hallan 2008</xref>, <xref ref-type="bibr" rid="B16">Fujita 2010</xref>) describe between four and seven species of <italic><named-content content-type="taxon-name">Lucanus</named-content></italic> in western Palaearctic: i.e. <italic><named-content content-type="taxon-name">Lucanus (Lucanus) cervus</named-content></italic> (Linnaeus, 1758), <italic><named-content content-type="taxon-name">Lucanus (Lucanus) ibericus</named-content></italic> Motschulsky, 1845, <italic><named-content content-type="taxon-name">Lucanus (Lucanus) orientalis</named-content></italic> Kraatz, 1860, <italic><named-content content-type="taxon-name">Lucanus (Lucanus) tetraodon</named-content></italic> Thunberg, 1806, <italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) barbarossa</named-content></italic> Fabricius, 1801, <italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) busignyi</named-content></italic> Planet, 1909 and <italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) macrophyllus</named-content></italic> Kraatz, 1860.</p><p>The distribution of many of these taxa remains poorly resolved, however, we can consider some of them as endangered. The practice of removing old trees and dead wood in past and current forest management, has had detrimental effects on this group of saproxylic beetles (<xref ref-type="bibr" rid="B33">Jansson and Coskun 2008</xref>, <xref ref-type="bibr" rid="B45">Nieto and Alexander 2010</xref>). Con<pmc-comment>PageBreak</pmc-comment>sequently, the loss of habitat might have reduced the range of some taxa, especially the Mediterranean taxa where deforestation started a few millennia ago (<xref ref-type="bibr" rid="B33">Jansson and Coskun 2008</xref>, <xref ref-type="bibr" rid="B7">Buse et al. 2010</xref>). At least <italic><named-content content-type="taxon-name">Lucanus cervus cervus</named-content></italic> seems to be able to cope with urbanisation (<xref ref-type="bibr" rid="B59">Thomaes et al. 2008</xref>) as long as the habitat turnover allows recolonisation (<xref ref-type="bibr" rid="B58">Thomaes 2009</xref>). In addition, beetle collecting can be considered as a threat when it goes hand in hand with large scale habitat destruction or when species rarity causes overexploitation (<xref ref-type="bibr" rid="B28">Holden 2007</xref>, <xref ref-type="bibr" rid="B61">Tournant et al. 2012</xref>). Another possible consequence of the international stag beetle trade is the introduction of non-native specimens which may cause genetic introgression (<xref ref-type="bibr" rid="B18">Goka et al. 2004</xref>) and transmission of parasites potentially pathogenic to native stag beetles (cf. <xref ref-type="bibr" rid="B18">Goka et al. 2004</xref>, <xref ref-type="bibr" rid="B34">Kanzaki et al. 2011</xref>). Unfortunately, legal protection is often missing or inadequate. The widely distributed <italic><named-content content-type="taxon-name">Lucanus cervus cervus</named-content></italic> is protected by the Habitats Directive of the European Union from 1992 (<xref ref-type="bibr" rid="B38">Luce 1996</xref>) and is listed as “near threatened” in the Red Data list of Europe (<xref ref-type="bibr" rid="B45">Nieto and Alexander 2010</xref>). <italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) barbarossa</named-content></italic> and <italic><named-content content-type="taxon-name">Lucanus tetraodon</named-content></italic> are mentioned in the IUCN list, but are rated “of least concern” (<xref ref-type="bibr" rid="B32">IUCN 2012</xref>), while <italic><named-content content-type="taxon-name">Lucanus ibericus</named-content></italic> is considered to be “vulnerable” within the EU 27 (<xref ref-type="bibr" rid="B45">Nieto and Alexander 2010</xref>).</p><p>More detailed information on the distribution and ecology of this species group is needed to get a clear view on their conservation status. But unless the problem of identification of European and West Asian <italic><named-content content-type="taxon-name">Lucanus</named-content></italic> is solved, it becomes difficult to set specific conservation priorities, without which rare, neglected and endangered species or Evolutionarily Significant Units (ESUs) may be unrecognised and thus, not given adequate conservation prioritisation (<xref ref-type="bibr" rid="B50">Ryder 1986</xref>, <xref ref-type="bibr" rid="B62">Waples 1991</xref>, <xref ref-type="bibr" rid="B42">Moritz 1994a</xref>, <xref ref-type="bibr" rid="B43">Moritz 1994b</xref>, <xref ref-type="bibr" rid="B15">Fraser and Bernatchez 2001</xref>). Molecular tools could help identification of stag beetles. The mitochondrial cytochrome <italic>c</italic> oxidase subunit I (COI) is the most widely used gene in barcoding animals (<xref ref-type="bibr" rid="B26">Hebert et al. 2003</xref>). The barcoding practice entails the analysis of the DNA sequence of a part of this mitochondrial gene, typically between 600 and 900 bp. In this study, we investigated the use of the 3’ end of the COI gene, different from the standard barcoding region, for the identification of western Palaearctic <italic><named-content content-type="taxon-name">Lucanus</named-content></italic> species and subspecies.</p></sec><sec sec-type="materials|methods"><title>Material and methods</title><sec sec-type="Taxonomy and morphology"><title>Taxonomy and morphology</title><pmc-comment>PageBreak</pmc-comment>
<p><italic><named-content content-type="taxon-name">Lucanus cervus</named-content></italic> has the widest geographical distribution in the genus and is very variable in form, size and colour (<xref ref-type="bibr" rid="B25">Harvey et al. 2011</xref>). Many subdivisions (i.e. subspecies or morphotypes) have been proposed and discussed. <italic><named-content content-type="taxon-name">Lucanus cervus cervus</named-content></italic> (Linnaeus, 1758), the main subspecies found throughout Europe, has, in general, four lamellae on the antennal clubs and is typically bicoloured (black head and thorax, and reddish brown elytra and mandibles). <italic><named-content content-type="taxon-name">Lucanus cervus akbesianus</named-content></italic> Planet, 1896 with generally six lamellae and large mandibles with a very open apical fork, inhabits southern Turkey and Syria. <italic><named-content content-type="taxon-name">Lucanus cervus turcicus</named-content></italic> Sturm, 1843 also has a six lamellate club, but its mandibles are comparable to <italic><named-content content-type="taxon-name">Lucanus cervus cervus</named-content></italic>. It is reported in Greece, Bulgaria and Trakya (European part of Turkey). Furthermore, <italic><named-content content-type="taxon-name">Lucanus cervus judaicus</named-content></italic> Planet, 1902 with a four lamellate club and reddish brown colour, is found in the more eastern parts of Turkey and in northern Syria. <italic><named-content content-type="taxon-name">Lucanus cervus laticornis</named-content></italic> Deyrolle, 1864, found in central and southern Turkey, has six long lamellae and the inner denticle of the mandibles is followed by two or three denticles. <italic><named-content content-type="taxon-name">Lucanus cervus fabiani</named-content></italic> Mulsant & Godart, 1855 is an endemic taxon inhabiting southern France and shows a five lamellate club and slender, slightly curved mandibles with a simple apex and post-median denticle along with a few other denticles. The taxa <italic><named-content content-type="taxon-name">Lucanus fabiani</named-content></italic> and <italic><named-content content-type="taxon-name">Lucanus pentaphyllus</named-content></italic> Reiche, 1853 are listed as synonyms of <italic><named-content content-type="taxon-name">Lucanus cervus cervus</named-content></italic> by <xref ref-type="bibr" rid="B4">Bartolozzi and Sprecher-Uebersax (2006)</xref>, but <italic><named-content content-type="taxon-name">Lucanus fabiani</named-content></italic> could well be considered as a valid species according to Boucher (unpublished data) while <italic><named-content content-type="taxon-name">Lucanus pentaphyllus</named-content></italic> may represent a small form of <italic><named-content content-type="taxon-name">Lucanus cervus</named-content></italic> with five lamellate clubs, a character that can also be found in <italic><named-content content-type="taxon-name">Lucanus cervus cervus</named-content></italic>. Other taxa [<italic><named-content content-type="taxon-name">Lucanus tauricus</named-content></italic> Motschulsky, 1845 (described from Crimea), <italic><named-content content-type="taxon-name">Lucanus poujadei</named-content></italic> Planet, 1897 (Kurdistan), <italic><named-content content-type="taxon-name">Lucanus mediadonta</named-content></italic> Lacroix, 1978 (Georgia) and <italic><named-content content-type="taxon-name">Lucanus pontbrianti</named-content></italic> Mulsant, 1839 (France)], recognised by some authors as valid subspecies or simple synonyms, were not included in this study. <xref ref-type="bibr" rid="B4">Bartolozzi and Sprecher-Uebersax (2006)</xref> only list <italic><named-content content-type="taxon-name">Lucanus cervus</named-content></italic> and <italic><named-content content-type="taxon-name">Lucanus judaicus</named-content></italic> as separate subspecies. <xref ref-type="bibr" rid="B23">Hallan (2008)</xref> adds <italic>akbesianus, fabiani, mediadonta, tauricus</italic> and <italic><named-content content-type="taxon-name">Lucanus turcicus</named-content></italic>, while <xref ref-type="bibr" rid="B36">Krajcik (2001)</xref> further includes <italic><named-content content-type="taxon-name">Lucanus pontbrianti</named-content></italic> and <italic><named-content content-type="taxon-name">Lucanus laticornis</named-content></italic>, although <xref ref-type="bibr" rid="B53">Schenk and Fiedler (2011)</xref> perceived <italic><named-content content-type="taxon-name">Lucanus laticornis</named-content></italic> as a separate species. On the other hand, <xref ref-type="bibr" rid="B12">Didier and Séguy (1953)</xref> also list <italic><named-content content-type="taxon-name">Lucanus capreolus</named-content></italic> Fuessly, 1775 (considered a small form of <italic><named-content content-type="taxon-name">Lucanus cervus</named-content></italic>) and <italic><named-content content-type="taxon-name">Lucanus poujadei</named-content></italic> while <xref ref-type="bibr" rid="B16">Fujita (2010)</xref> only recognises <italic><named-content content-type="taxon-name">Lucanus poujadei</named-content></italic> but does not list <italic><named-content content-type="taxon-name">Lucanus tauricus</named-content></italic> and <italic><named-content content-type="taxon-name">Lucanus mediadonta</named-content></italic> or the [<italic>pentaphyllus + fabiani + pontbrianti</italic>] complex.</p><p><italic><named-content content-type="taxon-name">Lucanus ibericus</named-content></italic> can be found from Albania to Iran and is sometimes considered a synonym of <italic><named-content content-type="taxon-name">Lucanus orientalis</named-content></italic>. Unlike <italic><named-content content-type="taxon-name">Lucanus cervus</named-content></italic>, <italic><named-content content-type="taxon-name">Lucanus ibericus</named-content></italic> is entirely reddish brown, has a pronotum without a smooth discal line, but with a sinuate posterior and distinct toothed posterior angles (non-sinuate pronotum and blunt angles in <italic><named-content content-type="taxon-name">Lucanus cervus</named-content></italic>). The mandibles of the males, which are shorter than those of a typical male <italic><named-content content-type="taxon-name">Lucanus cervus</named-content></italic> of equal size, can have an apex with two equal teeth or with the inner tooth fainted and a large internal denticle in the middle. In addition, <italic><named-content content-type="taxon-name">Lucanus ibericus</named-content></italic> has six, rarely five, long lamellae on the antennal club.</p><p><italic><named-content content-type="taxon-name">Lucanus tetraodon</named-content></italic> described from France, Italy, North Africa, Albania and Greece, can be perceived as a central Mediterranean species. In contrast to <italic><named-content content-type="taxon-name">Lucanus cervus</named-content></italic> and <italic><named-content content-type="taxon-name">Lucanus ibericus</named-content></italic>, the basal denticle of the mandibles of <italic><named-content content-type="taxon-name">Lucanus tetraodon</named-content></italic> is placed in the lower half. Like <italic><named-content content-type="taxon-name">Lucanus ibericus</named-content></italic>, the pronotal sides have sharp posterior angles, but the pronotal disc misses the central smooth line. <italic><named-content content-type="taxon-name">Lucanus tetraodon</named-content></italic> has six, occasionally five, lamellae on the antennal club. <italic><named-content content-type="taxon-name">Lucanus tetraodon</named-content></italic> is by some authors subdivided in subspecies <italic><named-content content-type="taxon-name">Lucanus tetraodon argeliensis</named-content></italic> Maes, 1995 in North Africa, <italic><named-content content-type="taxon-name">Lucanus tetraodon provincialis</named-content></italic> Colas, 1949 in South France, <italic><named-content content-type="taxon-name">Lucanus tetraodon corsicus</named-content></italic> Gautier des Cottes, 1860 in Corsica, <italic><named-content content-type="taxon-name">Lucanus tetraodon sicilianus</named-content></italic> Planet, 1899 in Sicily and finally <italic><named-content content-type="taxon-name">Lucanus tetraodon tetraodon</named-content></italic> Thunberg, 1806 elsewhere. In addition, specimens of problematic populations of <italic><named-content content-type="taxon-name">Lucanus cervus</named-content></italic> from a series of localities in central Italy (northern Latium and Umbria), are known to exhibit apparently intermediate morphological characters between <italic><named-content content-type="taxon-name">Lucanus cervus</named-content></italic> and <italic><named-content content-type="taxon-name">Lucanus tetraodon</named-content></italic>, which are sympatric in these areas (<xref ref-type="bibr" rid="B51">Santoro et al. 2009</xref>).</p><pmc-comment>PageBreak</pmc-comment>
<p>The <italic><named-content content-type="taxon-name">Pseudolucanus</named-content></italic> species all have six long lamellae forming the antennal club, their body is stout and entirely reddish or blackish brown. Included in this study are <italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) barbarossa</named-content></italic> from the Iberian peninsula and the Maghreb, and <italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) macrophyllus</named-content></italic> reported in south-west Turkey. <xref ref-type="bibr" rid="B36">Krajcik (2001)</xref> and <xref ref-type="bibr" rid="B23">Hallan (2008)</xref> list the latter as a subspecies of <italic><named-content content-type="taxon-name">Lucanus ibericus</named-content></italic>. <xref ref-type="bibr" rid="B53">Schenk and Fiedler (2011)</xref> recently quoted populations of <italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) busignyi</named-content></italic> in western Turkey, but this taxon is not included in this study.</p></sec><sec sec-type="Taxon sampling and DNA extraction"><title>Taxon sampling and DNA extraction</title><p>A large number of entomologists was contacted to obtain material from the different taxa and from different regions. The samples included whole beetles, especially in regions where identification is problematic, as well as parts of a beetle, sometimes found as road kill or as prey leftovers from birds. Samples were dried and kept at room temperature or preserved in absolute ethanol. In total 76 samples were collected. The species identification was performed, using comparative material and available identification keys. Six samples from Israel and Lebanon could not be identified to species. These unidentified <italic><named-content content-type="taxon-name">Lucanus</named-content></italic> specimens have a shape resembling in general the medium to small males of <italic><named-content content-type="taxon-name">Lucanus cervus akbesianus</named-content></italic> but with a mandibular structure similar to that of <italic><named-content content-type="taxon-name">Lucanus cervus turcicus</named-content></italic> (Zilioli et al. unpublished data). The tissue samples used for DNA extraction depended on what was available, but were mostly legs, which contain large muscles and are therefore rich in mitochondrial DNA (mtDNA). DNA was extracted from ground samples with the E.Z.N.A.® Forensic DNA Kit (Omega Bio-Tek), except for samples K1 and U6 (<xref ref-type="table" rid="T1">Table 1</xref>) from which DNA was extracted following the salting out procedure described by <xref ref-type="bibr" rid="B1">Aljanabi and Martinez (1997)</xref>. The integrity of the extracted DNA was checked spectrophotometrically on a ND-1000 Nano-Drop (NanoDrop Technologies) and its quality on 1% agarose gels.</p><table-wrap id="T1" orientation="portrait" position="float"><label>Table 1.</label><caption><p>List of samples included in the analysis. Primers used are denoted with ‘1’: C1-J-2183 and TL2-N-3014; ‘2’: LCint1F, LCint2F, LCint3F and LCint4F (for sample SB6 also the reverse primers were used); ‘3’: F - 5’ GGGGCATCAGTAGACCTAGC 3’ and R – 5’ TTCAGCAGGTGGTATTAGTTGG 3’.</p></caption><table frame="hsides" rules="groups"><tbody><tr><th rowspan="1" colspan="1">Species / subspecies</th><th rowspan="1" colspan="1">Code</th><th rowspan="1" colspan="1">Primers</th><th rowspan="1" colspan="1">Haplotype</th><th rowspan="1" colspan="1">GenBank acc. no.</th><th rowspan="1" colspan="1">Country</th><th rowspan="1" colspan="1">Latitude</th><th rowspan="1" colspan="1">Longitude</th><th rowspan="1" colspan="1">Date of sampling</th><th rowspan="1" colspan="1">Type of conservation</th><th rowspan="1" colspan="1">Gender</th></tr><tr><td rowspan="8" colspan="1"><italic><named-content content-type="taxon-name">Lucanus cervus akbesianus</named-content></italic></td><td rowspan="1" colspan="1">UA1</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">UA1</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737127">KF737127</ext-link></td><td rowspan="1" colspan="1">Turkey</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">37.721833, 30.828278</named-content></td><td rowspan="1" colspan="1">Jun 2010</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Female</td></tr><tr><td rowspan="1" colspan="1">UA2</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">UA2</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737128">KF737128</ext-link></td><td rowspan="1" colspan="1">Turkey</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">37.721833, 30.828278</named-content></td><td rowspan="1" colspan="1">Jun 2010</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1">UA3</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">UA3</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737129">KF737129</ext-link></td><td rowspan="1" colspan="1">Turkey</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">37.721833, 30.828278</named-content></td><td rowspan="1" colspan="1">Jun 2010</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1">UA4</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">UA4</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737130">KF737130</ext-link></td><td rowspan="1" colspan="1">Turkey</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">37.676200, 35.862100</named-content></td><td rowspan="1" colspan="1">2010</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1">UA5</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">UA5</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737131">KF737131</ext-link></td><td rowspan="1" colspan="1">Turkey</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">37.676200, 35.862100</named-content></td><td rowspan="1" colspan="1">2010</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1">UX1</td><td rowspan="1" colspan="1">2</td><td rowspan="1" colspan="1">UX1</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737132">KF737132</ext-link></td><td rowspan="1" colspan="1">Turkey</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">36.900000, 31.000000</named-content></td><td rowspan="1" colspan="1">Jun 2010</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1">U10</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">U10</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737125">KF737125</ext-link></td><td rowspan="1" colspan="1">Turkey</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">37.721833, 30.828278</named-content></td><td rowspan="1" colspan="1">Jun 2010</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1">U11</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">U10</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737126">KF737126</ext-link></td><td rowspan="1" colspan="1">Turkey</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">37.721833, 30.828278</named-content></td><td rowspan="1" colspan="1">Jun 2010</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="17" colspan="1"><italic><named-content content-type="taxon-name">Lucanus cervus cervus</named-content></italic></td><td rowspan="1" colspan="1">A1</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">A1</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737071">KF737071</ext-link></td><td rowspan="1" colspan="1">Belgium</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">50.772652, 4.537656</named-content></td><td rowspan="1" colspan="1">Jul 2008</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1">A3</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">A3</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737072">KF737072</ext-link></td><td rowspan="1" colspan="1">Belgium</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">50.736622, 4.331784</named-content></td><td rowspan="1" colspan="1">Jun 2009</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Female</td></tr><tr><td rowspan="1" colspan="1">C1</td><td rowspan="1" colspan="1">2</td><td rowspan="1" colspan="1">C1</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737093">KF737093</ext-link></td><td rowspan="1" colspan="1">Czech rep.</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">48.797935, 16.803576</named-content></td><td rowspan="1" colspan="1">May 2009</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1">D13</td><td rowspan="1" colspan="1">2</td><td rowspan="1" colspan="1">A3</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737078">KF737078</ext-link></td><td rowspan="1" colspan="1">France</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">45.391800, 1.139310</named-content></td><td rowspan="1" colspan="1">Jul 2010</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1">D4</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">D4</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737088">KF737088</ext-link></td><td rowspan="1" colspan="1">France</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">43.458090, 1.431787</named-content></td><td rowspan="1" colspan="1">Aug 2010</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1">D22</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">D22</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737092">KF737092</ext-link></td><td rowspan="1" colspan="1">France</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">47.861145, 2.820327</named-content></td><td rowspan="1" colspan="1">2009</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Female</td></tr><tr><td rowspan="1" colspan="1">F12</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">A3</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737079">KF737079</ext-link></td><td rowspan="1" colspan="1">Greece</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">39.808333, 22.653889</named-content></td><td rowspan="1" colspan="1">Jun 2009</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Female</td></tr><tr><td rowspan="1" colspan="1">F16</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">F16</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737083">KF737083</ext-link></td><td rowspan="1" colspan="1">Greece</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">39.808333, 22.653889</named-content></td><td rowspan="1" colspan="1">Jun 2009</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Female</td></tr><tr><td rowspan="1" colspan="1">F23</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">F23</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737082">KF737082</ext-link></td><td rowspan="1" colspan="1">Greece</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">39.762333, 21.663281</named-content></td><td rowspan="1" colspan="1">Jun 2009</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1">G3</td><td rowspan="1" colspan="1">2</td><td rowspan="1" colspan="1">G3</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737081">KF737081</ext-link></td><td rowspan="1" colspan="1">Hungary</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">47.701586, 18.834592</named-content></td><td rowspan="1" colspan="1">Jul 2009</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Female</td></tr><tr><td rowspan="1" colspan="1">I2</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">I2</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737084">KF737084</ext-link></td><td rowspan="1" colspan="1">Italy</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">45.779241, 8.732981</named-content></td><td rowspan="1" colspan="1">Jun 2009</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1">I3</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">A3</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737080">KF737080</ext-link></td><td rowspan="1" colspan="1">Italy</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">45.779241, 8.732981</named-content></td><td rowspan="1" colspan="1">Jun 2009</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1">I4</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">I4</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737085">KF737085</ext-link></td><td rowspan="1" colspan="1">Italy</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">45.779241, 8.732981</named-content></td><td rowspan="1" colspan="1">Jun 2009</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1">N3</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">N3</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737086">KF737086</ext-link></td><td rowspan="1" colspan="1">Portugal</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">38.795900, -9.397390</named-content></td><td rowspan="1" colspan="1">Jul 2010</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1">O9</td><td rowspan="1" colspan="1">3</td><td rowspan="1" colspan="1">O9</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737087">KF737087</ext-link></td><td rowspan="1" colspan="1">Romania</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">47.102400, 24.450700</named-content></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">S15</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">S15</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737094">KF737094</ext-link></td><td rowspan="1" colspan="1">Spain</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">40.385100, -6.608460</named-content></td><td rowspan="1" colspan="1">Aug 2009</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1">S19</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">A3</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737076">KF737076</ext-link></td><td rowspan="1" colspan="1">Spain</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">43.304009, -4.814970</named-content></td><td rowspan="1" colspan="1">Jul 2009</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Female</td></tr><tr><td rowspan="5" colspan="1"></td><td rowspan="1" colspan="1">V2</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">A3</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737077">KF737077</ext-link></td><td rowspan="1" colspan="1">UK</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">52.028936, 1.067369</named-content></td><td rowspan="1" colspan="1">Aug 2009</td><td rowspan="1" colspan="1">dried</td><td rowspan="1" colspan="1">Female</td></tr><tr><td rowspan="1" colspan="1">V26</td><td rowspan="1" colspan="1">3</td><td rowspan="1" colspan="1">V26</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737091">KF737091</ext-link></td><td rowspan="1" colspan="1">UK</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">50.966300, -0.209294</named-content></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">V44</td><td rowspan="1" colspan="1">3</td><td rowspan="1" colspan="1">V44</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737089">KF737089</ext-link></td><td rowspan="1" colspan="1">UK</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">51.260100, 0.844280</named-content></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">W9</td><td rowspan="1" colspan="1">2</td><td rowspan="1" colspan="1">W9</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737090">KF737090</ext-link></td><td rowspan="1" colspan="1">Ukraine</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">49.826900, 36.325800</named-content></td><td rowspan="1" colspan="1">Jun 2007</td><td rowspan="1" colspan="1">dried</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1">X1</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1">X1</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/FJ606555">FJ606555</ext-link></td><td rowspan="1" colspan="1">France</td><td rowspan="1" colspan="5">(<xref ref-type="bibr" rid="B37">Lin et al. 2011</xref>)</td></tr><tr><td rowspan="1" colspan="1"><italic><named-content content-type="taxon-name">Lucanus cervus fabiani</named-content></italic></td><td rowspan="1" colspan="1">D11</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">D11</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737121">KF737121</ext-link></td><td rowspan="1" colspan="1">France</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">43.195300, 5.753740</named-content></td><td rowspan="1" colspan="1">Jun 2010</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1"><italic><named-content content-type="taxon-name">Lucanus cervus judaicus</named-content></italic></td><td rowspan="1" colspan="1">UJ1</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">UJ1</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737112">KF737112</ext-link></td><td rowspan="1" colspan="1">Turkey</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">37.068100, 36.261600</named-content></td><td rowspan="1" colspan="1">Jul 2010</td><td rowspan="1" colspan="1">dried</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="2" colspan="1"><italic><named-content content-type="taxon-name">Lucanus cervus laticornis</named-content></italic></td><td rowspan="1" colspan="1">UL2</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">UL2</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737119">KF737119</ext-link></td><td rowspan="1" colspan="1">Turkey</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">36.875669, 30.457431</named-content></td><td rowspan="1" colspan="1">Jun 2007</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1">UL3</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">UL3</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737120">KF737120</ext-link></td><td rowspan="1" colspan="1">Turkey</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">37.763600, 30.558900</named-content></td><td rowspan="1" colspan="1">1995</td><td rowspan="1" colspan="1">dried</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="4" colspan="1"><italic><named-content content-type="taxon-name">Lucanus cervus pentaphyllus</named-content></italic></td><td rowspan="1" colspan="1">C2</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">A3</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737075">KF737075</ext-link></td><td rowspan="1" colspan="1">Czech rep.</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">48.797935, 16.803576</named-content></td><td rowspan="1" colspan="1">May 2009</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1">F13</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">F13</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737104">KF737104</ext-link></td><td rowspan="1" colspan="1">Greece</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">39.808333, 22.653889</named-content></td><td rowspan="1" colspan="1">Jun 2009</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Female</td></tr><tr><td rowspan="1" colspan="1">I1</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">A3</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737073">KF737073</ext-link></td><td rowspan="1" colspan="1">Italy</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">45.779241, 8.732981</named-content></td><td rowspan="1" colspan="1">Jun 2009</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1">W7</td><td rowspan="1" colspan="1">2</td><td rowspan="1" colspan="1">A3</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737074">KF737074</ext-link></td><td rowspan="1" colspan="1">Ukraine</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">48.950200, 38.497600</named-content></td><td rowspan="1" colspan="1">Jul 2002</td><td rowspan="1" colspan="1">dried</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="13" colspan="1"><italic><named-content content-type="taxon-name">Lucanus cervus turcicus</named-content></italic></td><td rowspan="1" colspan="1">B1</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">B1</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737096">KF737096</ext-link></td><td rowspan="1" colspan="1">Bulgaria</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">42.162733, 27.737650</named-content></td><td rowspan="1" colspan="1">Jul 2009</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1">B2</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">B2</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737098">KF737098</ext-link></td><td rowspan="1" colspan="1">Bulgaria</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">41.407800, 25.578583</named-content></td><td rowspan="1" colspan="1">Jul 2009</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1">B7</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">B7</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737099">KF737099</ext-link></td><td rowspan="1" colspan="1">Bulgaria</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">42.060792, 27.977000</named-content></td><td rowspan="1" colspan="1">Jul 2009</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1">B9</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">B1</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737097">KF737097</ext-link></td><td rowspan="1" colspan="1">Bulgaria</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">42.120183, 27.900405</named-content></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">F15</td><td rowspan="1" colspan="1">2</td><td rowspan="1" colspan="1">F15</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737105">KF737105</ext-link></td><td rowspan="1" colspan="1">Greece</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">39.808333, 22.653889</named-content></td><td rowspan="1" colspan="1">Jun 2009</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1">F7</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">F7</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737107">KF737107</ext-link></td><td rowspan="1" colspan="1">Greece</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">39.866667, 22.733333</named-content></td><td rowspan="1" colspan="1">Jun 2009</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">F8</td><td rowspan="1" colspan="1">2</td><td rowspan="1" colspan="1">F7</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737108">KF737108</ext-link></td><td rowspan="1" colspan="1">Greece</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">39.866667, 22.733333</named-content></td><td rowspan="1" colspan="1">Jun 2009</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">F9</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">F9</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737106">KF737106</ext-link></td><td rowspan="1" colspan="1">Greece</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">39.808333, 22.653889</named-content></td><td rowspan="1" colspan="1">Jun 2009</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Female</td></tr><tr><td rowspan="1" colspan="1">F11</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">F11</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737100">KF737100</ext-link></td><td rowspan="1" colspan="1">Greece</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">39.808333, 22.653889</named-content></td><td rowspan="1" colspan="1">Jun 2009</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1">F17</td><td rowspan="1" colspan="1">2</td><td rowspan="1" colspan="1">F17</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737101">KF737101</ext-link></td><td rowspan="1" colspan="1">Greece</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">39.808333, 22.653889</named-content></td><td rowspan="1" colspan="1">Jun 2009</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">F20</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">F20</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737102">KF737102</ext-link></td><td rowspan="1" colspan="1">Greece</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">39.808333, 22.653889</named-content></td><td rowspan="1" colspan="1">Jun 2009</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1">F21</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">F21</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737103">KF737103</ext-link></td><td rowspan="1" colspan="1">Greece</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">39.808333, 22.653889</named-content></td><td rowspan="1" colspan="1">Jun 2009</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1">U3</td><td rowspan="1" colspan="1">2</td><td rowspan="1" colspan="1">U3</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737109">KF737109</ext-link></td><td rowspan="1" colspan="1">Turkey</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">41.800000, 27.950000</named-content></td><td rowspan="1" colspan="1">Jul 2009</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="6" colspan="1">Unknown species of <italic><named-content content-type="taxon-name">Lucanus</named-content></italic></td><td rowspan="1" colspan="1">H1</td><td rowspan="1" colspan="1">2</td><td rowspan="1" colspan="1">H1</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737116">KF737116</ext-link></td><td rowspan="1" colspan="1">Israel</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">33.217100, 35.753500</named-content></td><td rowspan="1" colspan="1">Aug 2009</td><td rowspan="1" colspan="1">ethanol (after freezing)</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1">H2</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">H2</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737113">KF737113</ext-link></td><td rowspan="1" colspan="1">Israel</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">33.217100, 35.753500</named-content></td><td rowspan="1" colspan="1">Aug 2009</td><td rowspan="1" colspan="1">dried</td><td rowspan="1" colspan="1">Female</td></tr><tr><td rowspan="1" colspan="1">H3</td><td rowspan="1" colspan="1">2</td><td rowspan="1" colspan="1">H3</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737117">KF737117</ext-link></td><td rowspan="1" colspan="1">Israel</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">33.217100, 35.753500</named-content></td><td rowspan="1" colspan="1">Jul 2009</td><td rowspan="1" colspan="1">dried</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1">H4</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">H4</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737114">KF737114</ext-link></td><td rowspan="1" colspan="1">Israel</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">33.217100, 35.753500</named-content></td><td rowspan="1" colspan="1">Jul 2009</td><td rowspan="1" colspan="1">dried</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1">H5</td><td rowspan="1" colspan="1">2</td><td rowspan="1" colspan="1">H5</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737115">KF737115</ext-link></td><td rowspan="1" colspan="1">Israel</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">32.959600, 35.864500</named-content></td><td rowspan="1" colspan="1">1998</td><td rowspan="1" colspan="1">dried</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1">J2<xref ref-type="fn" rid="T1TN1">†</xref></td><td rowspan="1" colspan="1">2</td><td rowspan="1" colspan="1">J2</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737118">KF737118</ext-link></td><td rowspan="1" colspan="1">Lebanon</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1">Jul 2009</td><td rowspan="1" colspan="1">dried</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1"><italic><named-content content-type="taxon-name">Lucanus ibericus</named-content></italic></td><td rowspan="1" colspan="1">U6</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">U6</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737110">KF737110</ext-link></td><td rowspan="1" colspan="1">Turkey</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">40.290300, 38.424200</named-content></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1"><italic><named-content content-type="taxon-name">Lucanus tetraodon provincialis</named-content></italic></td><td rowspan="1" colspan="1">D6</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">D6</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737111">KF737111</ext-link></td><td rowspan="1" colspan="1">France</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">43.066700, 5.850000</named-content></td><td rowspan="1" colspan="1">Jun 2010</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1"><italic><named-content content-type="taxon-name">Lucanus tetraodon</named-content></italic></td><td rowspan="1" colspan="1">X2</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1">X2</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/EF487727">EF487727</ext-link></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="5">(<xref ref-type="bibr" rid="B31">Hunt et al. 2007</xref>)</td></tr><tr><td rowspan="3" colspan="1"><italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) barbarossa</named-content></italic></td><td rowspan="1" colspan="1">SB1</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">SB1</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737122">KF737122</ext-link></td><td rowspan="1" colspan="1">Spain</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">40.828139, -3.831811</named-content></td><td rowspan="1" colspan="1">Jul 2004</td><td rowspan="1" colspan="1">dried, later on ethanol</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1">SB6<xref ref-type="fn" rid="T1TN1">†</xref></td><td rowspan="1" colspan="1">2</td><td rowspan="1" colspan="1">SB6</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737124">KF737124</ext-link></td><td rowspan="1" colspan="1">Spain</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">41.067361, -3.585322</named-content></td><td rowspan="1" colspan="1">Sep 2010</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Female</td></tr><tr><td rowspan="1" colspan="1">SB7</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">SB7</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737123">KF737123</ext-link></td><td rowspan="1" colspan="1">Spain</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">36.885000, -3.982000</named-content></td><td rowspan="1" colspan="1">May 2010</td><td rowspan="1" colspan="1">ethanol</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="1" colspan="1"><italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) macrophyllus</named-content></italic></td><td rowspan="1" colspan="1">UB1<xref ref-type="fn" rid="T1TN1">†</xref></td><td rowspan="1" colspan="1">2</td><td rowspan="1" colspan="1">UB1</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737095">KF737095</ext-link></td><td rowspan="1" colspan="1">Turkey</td><td rowspan="1" colspan="2"><named-content content-type="dwc:verbatimCoordinates">36.501944, 33.089167</named-content></td><td rowspan="1" colspan="1">Aug 2006</td><td rowspan="1" colspan="1">dried</td><td rowspan="1" colspan="1">Male</td></tr><tr><td rowspan="2" colspan="1"><italic><named-content content-type="taxon-name">Dorcus parallelipipedus</named-content></italic></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">K1</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737133">KF737133</ext-link></td><td rowspan="1" colspan="1">Montenegro</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1">X3</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/DQ156023">DQ156023</ext-link></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="5">(<xref ref-type="bibr" rid="B31">Hunt et al. 2007</xref>)</td></tr><tr><td rowspan="7" colspan="1"><italic><named-content content-type="taxon-name">Lucanus formosanus</named-content></italic></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1">X4</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/FJ606632">FJ606632</ext-link></td><td rowspan="1" colspan="1"></td><td rowspan="7" colspan="5">(<xref ref-type="bibr" rid="B29">Huang and Lin 2010</xref>)</td></tr><tr><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1">X5</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/FJ606630">FJ606630</ext-link></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1">X6</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/FJ606628">FJ606628</ext-link></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1">X5</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/FJ606626">FJ606626</ext-link></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1">X5</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/FJ606624">FJ606624</ext-link></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1">X5</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/FJ606622">FJ606622</ext-link></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1">X8</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/FJ606583">FJ606583</ext-link></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1"><italic><named-content content-type="taxon-name">Lucanus hermani</named-content></italic></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1">X9</td><td rowspan="1" colspan="1"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/FJ606552">FJ606552</ext-link></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="5">(<xref ref-type="bibr" rid="B37">Lin et al. 2011</xref>)</td></tr></tbody></table><table-wrap-foot><fn id="T1TN1"><p><bold>†</bold> sequences with a maximum of seven double peaks.</p></fn></table-wrap-foot></table-wrap></sec><sec sec-type="Sequencing"><title>Sequencing</title><p>We first attempted to sequence the COI barcoding region with the primers developed by Folmer et al. (1994) on a subset of samples. Despite PCR optimization trials, amplification of this fragment largely failed. Instead, a 800 bp fragment of the 3’ end of the COI gene was amplified using the primer set C1-J-2183 (5’ CAACATTTATTTTGATTTTTTGG 3’) and TL2-N-3014 (5’ TCCAATGCACTAATCTGCCATATTA 3’) (<xref ref-type="bibr" rid="B54">Simon et al. 1994</xref>). This fragment does not overlap with the standard barcoding region. For samples O9 and V44 (<xref ref-type="table" rid="T1">Table 1</xref>) we used species-specific primers (F - 5’ GGGGCATCAGTAGACCTAGC 3’ and R – 5’ TTCAGCAGGTGGTATTAGTTGG 3’), designed from sequences on GenBank and used to PCR amplify a 1089 bp stretch of the COI gene. Reactions were performed in total volumes of 40 µl containing 5.2 µl of 10 × Taq buffer with 500 mM KCl (Fermentas, Thermo Scientific), 3.12 µl of MgCl<sub>2</sub> (25 mM), 0.78 µl dNTP (10 mM), 2.08 µl of each <pmc-comment>PageBreak</pmc-comment><pmc-comment>PageBreak</pmc-comment><pmc-comment>PageBreak</pmc-comment><pmc-comment>PageBreak</pmc-comment>primer (10 µM), 0.8 U Taq DNA polymerase (Fermentas, Thermo Scientific), 26.42 µl sterile distilled water. 12 µl of diluted DNA (3.5–5 ng/ µl) was added. The temperature cycle was 94 °C for 1 min, then 5 cycles of 94 °C for 1 min, 45 °C for 1 min 30 s and 72 °C for 1 min and 30 s. This was followed by 40 cycles of 94 °C for 1 min, 50 °C for 1 min 30 s and 72 °C for 1 min, and finally a single cycle at 72 °C for 5 min. PCR products were cleaned enzymatically with DNA Clean & Concentrator<sup>TM</sup>-5 (Zymo Research). When samples failed to amplify, mostly dried or bad quality samples, internal primers were used to allow amplification of four overlapping fragments of about 250 bp within the same 3’ end of the COI gene: LCint1 (F – 5’ CTTCGGCCACCCAGAAGT 3’ and R – 5’ TCCAGTAGGAACAGCAATRAT 3’), LCint2 (F – 5’ CGAGCCTACTTCACATCAGC 3’ and R – 5’ GCAAAAACTGCACCTATTGAAA 3’), LCint3 (F – 5’ GCTCACTTCCATTATGTACTTTCAA 3’ and R – 5’ GAGAGCCAAATGATGAAATAATGTT 3’) and LCint4 (F – 5’ CCCTGATGCCTACACCACAT 3’ and R – 5’ CCAATGCACTAATCTGCCATA 3’). PCR amplification was performed in 2.6 µl of 10x Taq buffer with 500 mM KCl, 2.08 µl of MgCl2 (25 mM), 0.39 µl dNTP (10 mM), 2.6 µl of each primer (10 µM), 0.8 U Taq DNA polymerase (Fermentas, Thermo Scientific), 9.57 µl sterile distilled water, resulting in a total volume of 20 µl to which 6 µl of diluted DNA (3.5–5 ng/ µl) was added. The PCR reaction was then conducted with the following cycle: 94 °C for 3 min, then 45 cycles of 94 °C for 45 s, 59 °C for 45 s and 72 °C for 1 min 30 s, and finally a single cycle at 72 °C for 6 min. PCR products were checked on 2% agarose horizontal gels and purified using USB® ExoSAP-IT® (Isogen Life Science). DNA sequencing was performed by a commercial company (BaseClear, Leiden, the Netherlands) or on an automatic ABI 3500 Genetic Analyzer (Applied Biosystems). Both forward and reverse primers were used except when internal primers were used for PCR, in which case sequencing was performed using the respective forward primers (except for five samples of <italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) barbarossa</named-content></italic>, where both forward and reverse primers were used).</p><p>COI sequences available on GenBank were added. The COI sequence of <italic><named-content content-type="taxon-name">Lucanus cervus cervus</named-content></italic> obtained by <xref ref-type="bibr" rid="B37">Lin et al. (2011</xref>; GenBank acc. no. <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/FJ606555">FJ606555</ext-link>) was used as a reference for the subspecies with the highest number of specimens in this study. We selected two Asian stag beetle species, <italic><named-content content-type="taxon-name">Lucanus formosanus</named-content></italic> Planet, 1899 and <italic><named-content content-type="taxon-name">Lucanus hermani</named-content></italic> DeLisle, 1973, and <italic><named-content content-type="taxon-name">Dorcus parallelipipedus</named-content></italic> (Linnaeus, 1758) (lesser stag beetle; <named-content content-type="taxon-name">Lucanidae</named-content>) as outgroup species. Except for one available sample of the latter, the COI gene sequences of the taxa were obtained from GenBank (<italic><named-content content-type="taxon-name">Dorcus parallelipipedus</named-content></italic>: <xref ref-type="bibr" rid="B31">Hunt et al. 2007</xref>; GenBank acc. no. <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/DQ156023">DQ156023</ext-link>; <italic><named-content content-type="taxon-name">Lucanus formosanus</named-content></italic>: <xref ref-type="bibr" rid="B29">Huang and Lin 2010</xref>; GenBank acc. no. <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/FJ606632">FJ606632</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/FJ606630">FJ606630</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/FJ606628">FJ606628</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/FJ606626">FJ606626</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/FJ606624">FJ606624</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/FJ606622">FJ606622</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/FJ606583">FJ606583</ext-link>; <italic><named-content content-type="taxon-name">Lucanus hermani</named-content></italic>: <xref ref-type="bibr" rid="B37">Lin et al. 2011</xref>; GenBank acc. no.: <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/FJ606552">FJ606552</ext-link>). In the study of <xref ref-type="bibr" rid="B31">Hunt et al. (2007)</xref> the <named-content content-type="taxon-name">Dorcinae</named-content> formed a sisterclade of the <named-content content-type="taxon-name">Lucaninae</named-content>. Finally, part of the COI sequence of <italic><named-content content-type="taxon-name">Lucanus tetraodon</named-content></italic> obtained by <xref ref-type="bibr" rid="B32">Hunt et al. (2007</xref>; GenBank acc. no. <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/EF487727">EF487727</ext-link>) was used in addition to the sequence of <italic><named-content content-type="taxon-name">Lucanus tetraodon provincialis</named-content></italic>.</p><p>DNA sequences have been deposited in GenBank under accession numbers <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737071">KF737071</ext-link> to <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/KF737133">KF737133</ext-link> (<xref ref-type="table" rid="T1">Table 1</xref>).</p><pmc-comment>PageBreak</pmc-comment>
</sec><sec sec-type="Alignment and sequence quality control"><title>Alignment and sequence quality control</title><p>Overall quality of the sequences was evaluated manually. Only samples with high quality chromatograms for at least 300 bp were retained for further analyses. Sequences were aligned by hand and using CLUSTALW v1.4 (<xref ref-type="bibr" rid="B60">Thompson et al. 1994</xref>) in BIOEDIT v7.0.0 (<xref ref-type="bibr" rid="B22">Hall 1999</xref>). Sequences were trimmed to 670 bases. Duplicate haplotypes were removed using DUPLICATESFINDER v1.1 (<ext-link ext-link-type="uri" xlink:href="http://bioinfotutlets.blogspot.be/2009/09/duplicates-finder-java-standalone.html">http://bioinfotutlets.blogspot.be/2009/09/duplicates-finder-java-standalone.html</ext-link>). We searched for potential NUMTs (nuclear mitochondrial pseudogene sequences) or heteroplasmy by manually checking for the presence of double peaks and indels, and by looking for stop codons (<xref ref-type="bibr" rid="B56">Song et al. 2008</xref>, <xref ref-type="bibr" rid="B8">Calvignac et al. 2011</xref>) using MEGA c5.01 with the implemented invertebrate mtDNA genetic code to translate the sequences (<xref ref-type="bibr" rid="B57">Tamura et al. 2011</xref>). We only retained sequences with a maximum of 7 polymorphic positions, which were treated as ambiguities. Finally, we constructed a Neighbour-Joining (NJ) tree with MEGA5 using 10 000 bootstraps, based on Kimura’s 2-parameter distances (K2P) (<xref ref-type="bibr" rid="B35">Kimura 1980</xref>). For comparison, a Bayesian inference approach (BI) was used as well. The Bayesian analysis was conducted with MRBAYES v3.1.2 (<xref ref-type="bibr" rid="B30">Huelsenbeck and Ronquist 2001</xref>, <xref ref-type="bibr" rid="B49">Ronquist and Huelsenbeck 2003</xref>) under the GTR+I+G model, simulating 4 Monte Carlo Markov Chains (MCMC) for 2 000 000 generations each. Trees were sampled every 100 generations and the first 300 000 generations were excluded as burn-in. A consensus tree was constructed with posterior probabilities. The MRBAYES analyses were carried out on the Bioportal at Oslo University (<ext-link ext-link-type="uri" xlink:href="http://www.bioportal.uio.no">http://www.bioportal.uio.no</ext-link>). The GTR+I+G model used in MRBAYES is closely related to the TIM3+I+G model, which was selected by JMODELTEST v0.1.1 (<xref ref-type="bibr" rid="B19">Guindon and Gascuel 2003</xref>, <xref ref-type="bibr" rid="B48">Posada 2008</xref>) as the best-fit model under the Akaike information criterion (AIC).</p></sec><sec sec-type="Genetic distances and nucleotide diagnostics"><title>Genetic distances and nucleotide diagnostics</title><p>As K2P-distance is the most commonly used distance metric in DNA barcoding (<xref ref-type="bibr" rid="B26">Hebert et al. 2003</xref>), it was employed here for comparison. It allows to compare the behavior of the DNA fragment we used to the standard barcode region which is situated in the same gene. When possible, simple nucleotide diagnostics were identified for each (sub)species. If less than two simple nucleotide diagnostics were present (<xref ref-type="bibr" rid="B52">Sarkar et al. 2002</xref>), a compound diagnostic was detected using the algorithm of <xref ref-type="bibr" rid="B65">Wong et al. (2009)</xref>.</p></sec></sec><sec sec-type="Results"><title>Results</title><sec sec-type="Alignment and sequence quality"><title>Alignment and sequence quality</title><p>Of a total of 76 samples, thirteen samples with low quality sequences were removed: five <pmc-comment>PageBreak</pmc-comment><italic><named-content content-type="taxon-name">Lucanus cervus cervus</named-content></italic>, one <italic><named-content content-type="taxon-name">Lucanus cervus pentaphyllus</named-content></italic>, three <italic><named-content content-type="taxon-name">Lucanus cervus turcicus</named-content></italic> and four <italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) barbarossa</named-content></italic>. Three sequences showed a few double peaks: one <italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) barbarossa</named-content></italic> (SB6: 5 ambiguous sites), one <italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) macrophyllus</named-content></italic> (UB1: 7 ambiguous sites) and one unidentified species of <italic><named-content content-type="taxon-name">Lucanus</named-content></italic> (J2: 2 ambiguous sites) (<xref ref-type="table" rid="T1">Table 1</xref>). None exhibited indels or stop codons which are indicative of the presence of NUMTs (<xref ref-type="bibr" rid="B6">Buhay 2009</xref>). The remaining 63 samples and 11 sequences obtained from GenBank are listed in <xref ref-type="table" rid="T1">Table 1</xref>. The final alignment entailed 74 sequences, representing 60 haplotypes. Incomplete sequences were obtained for the following taxa: taxon H4 with 500 bp of which the reverse sequence failed and taxon J2 of which forward sequences of only the first and third smaller fragments could be produced, resulting in a total of 383 bp. Both taxa were specimens of the unidentified <italic><named-content content-type="taxon-name">Lucanus</named-content></italic> specimens (<xref ref-type="table" rid="T1">Table 1</xref>). Likewise, the sequence of <italic><named-content content-type="taxon-name">Lucanus tetraodon</named-content></italic> found in GenBank (named X2), was 122 bp short at the 3’ end. One other taxon, H3 (<italic><named-content content-type="taxon-name">Lucanus</named-content></italic> sp.) missed a mere 5 bp at the 5’ end.</p><p>Both the NJ tree and the BI tree showed the same overall configuration (<xref ref-type="fig" rid="F1">Figure 1</xref> and Appendix 1, respectively) except for the position of the unidentified <italic><named-content content-type="taxon-name">Lucanus</named-content></italic> specimens. In the NJ tree these specimens fall into two clusters with unresolved affinities (<xref ref-type="fig" rid="F1">Figure 1</xref>). In the BI tree they form a single well-supported clade together with specimens identified as <italic><named-content content-type="taxon-name">Lucanus cervus judaicus</named-content></italic> and <italic><named-content content-type="taxon-name">Lucanus cervus laticornis</named-content></italic> (Appendix 1). The unidentified specimens fail to form a single monophyletic cluster as one subclade also includes <italic><named-content content-type="taxon-name">Lucanus cervus judaicus</named-content></italic>. The BI tree showed <italic><named-content content-type="taxon-name">Lucanus cervus laticornis</named-content></italic> to be monophyletic with probability 1, instead of paraphyletic as was shown in the NJ tree with bootstrap support below 70%. In both trees, several species as well as subspecies fall into distinct clades, whereas <italic><named-content content-type="taxon-name">Lucanus cervus cervus</named-content></italic>, <italic><named-content content-type="taxon-name">Lucanus cervus turcicus</named-content></italic>, <italic><named-content content-type="taxon-name">Lucanus cervus pentaphyllus</named-content></italic>, <italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) macrophyllus</named-content></italic> and <italic><named-content content-type="taxon-name">Lucanus ibericus</named-content></italic> cluster in the same shallow clade (called the ‘<italic><named-content content-type="taxon-name">Lucanus cervus cervus</named-content></italic> clade’ hereafter). In addition, three out of four samples of <italic><named-content content-type="taxon-name">Lucanus cervus pentaphyllus</named-content></italic> share a haplotype with <italic><named-content content-type="taxon-name">Lucanus cervus cervus</named-content></italic> (haplotype A3) which is the most common haplotype among <italic><named-content content-type="taxon-name">Lucanus cervus</named-content></italic> sequences (<xref ref-type="table" rid="T1">Table 1</xref>). Within this clade <italic><named-content content-type="taxon-name">Lucanus cervus cervus</named-content></italic>, <italic><named-content content-type="taxon-name">Lucanus cervus turcicus</named-content></italic> and <italic><named-content content-type="taxon-name">Lucanus cervus pentaphyllus</named-content></italic> are polyphyletic. Unexpectedly, one sample of <italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) barbarossa</named-content></italic> and the sample of <italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) macrophyllus</named-content></italic> are also embedded in this clade. Looking at the sequences, they only differ from haplotype A3 at their five and seven ambiguous sites, respectively. Because the two other specimens of <italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) barbarossa</named-content></italic> form a separate clade with <italic><named-content content-type="taxon-name">Lucanus cervus fabiani</named-content></italic>, sample SB6 is excluded from further calculations but will be discussed below.</p><fig id="F1" orientation="portrait" position="float"><label>Figure 1.</label><caption><p>Bootstrap consensus NJ tree inferred from 10 000 replicates, with a cut off value of 70%, based on K2P-distances between 60 haplotypes of the 3’ end of the COI gene.</p></caption><graphic xlink:href="ZooKeys-365-105-g001"></graphic></fig></sec><sec sec-type="Genetic distances"><title>Genetic distances</title><p>The nucleotide composition of all the sequences was AT-rich, with 29.5% A, 35.2% T, 15.5% G and 19.7% C. There were 36.4% nucleotide sites variable and 12.1% variable amino acid sites, of which 94.3% and 77.8% were parsimony informative, respectively. When <italic><named-content content-type="taxon-name">Dorcus parallelipipedus</named-content></italic> was excluded from the dataset, variable sites decreased to 33.3% for nucleotides and 7.2% for amino acids (94.2% and 56.2% parsimony informative, respectively). Nucleotide composition and K2P-distances calculated for each codon position are shown in <xref ref-type="table" rid="T3">Table 3</xref>.</p><p>Although specimen J2 of the unidentified specimens of <pmc-comment>PageBreak</pmc-comment><pmc-comment>PageBreak</pmc-comment><italic><named-content content-type="taxon-name">Lucanus</named-content></italic> clustered with the other specimens of the same taxon in the NJ and BI trees, the pairwise interspecific K2P-distances with J2 differed substantially from those with H1 to H5 (comparisons with <italic><named-content content-type="taxon-name">Lucanus cervus judaicus</named-content></italic> not included). More specifically, the minimum pairwise interspecific K2P-distance between J2 and the other unidentified taxa was 0.064 opposed to 0.087–0.095 when taking H1 to H5 into account. J2 is one of three incomplete sequences and missing information from position 179 to 399 in the sequence of J2 where several simple nucleotide diagnostics are present (Appendix 2). Therefore, this sample was removed from the dataset for subsequent analysis.</p><p>The congeneric interspecific K2P-distances between the western Palaearctic taxa and the eastern Asian species <italic><named-content content-type="taxon-name">Lucanus formosanus</named-content></italic> and <italic><named-content content-type="taxon-name">Lucanus hermani</named-content></italic> range from 0.156 to 0.198. Distances between taxa of <italic><named-content content-type="taxon-name">Lucanus</named-content></italic> and <italic><named-content content-type="taxon-name">Dorcus</named-content></italic> went from 0.211 until 0.259. K2P-distances within and between the investigated western Palaearctic taxa of <italic><named-content content-type="taxon-name">Lucanus</named-content></italic> are shown in <xref ref-type="table" rid="T2">Table 2</xref>. As indicated by the NJ and BI trees, the taxa <italic><named-content content-type="taxon-name">Lucanus cervus cervus</named-content></italic>, <italic><named-content content-type="taxon-name">Lucanus cervus pentaphyllus</named-content></italic>, <italic><named-content content-type="taxon-name">Lucanus cervus turcicus</named-content></italic> and <italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) macrophyllus</named-content></italic> cannot be distinguished based on the COI fragment; K2P-distances range from 0 to 0.021, and all taxa are reciprocally polyphyletic. Whereas the first three subspecies of <italic><named-content content-type="taxon-name">Lucanus cervus</named-content></italic> are distinguished solely on the basis of the number of lamellae on the antennal club, <italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) macrophyllus</named-content></italic> is morphologically much more distinctive, resembling <italic><named-content content-type="taxon-name">Lucanus ibericus</named-content></italic>. Although <italic><named-content content-type="taxon-name">Lucanus ibericus</named-content></italic> is part of the <italic><named-content content-type="taxon-name">Lucanus cervus cervus</named-content></italic> clade, it shows slightly higher K2P-distances with the other members of this clade (0.028–0.032). Note that we only had a single specimen. Moderate to relatively high within (sub)species distances were found for <italic><named-content content-type="taxon-name">Lucanus cervus laticornis</named-content></italic> (0.085), certain specimens of the unidentified <italic><named-content content-type="taxon-name">Lucanus</named-content></italic> sp. (max. 0.054) and <italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) barbarossa</named-content></italic> (0.53). On the other hand, between the latter and <italic><named-content content-type="taxon-name">Lucanus cervus fabiani</named-content></italic> a small to moderate distance exists (0.004 and 0.058). This is also the case between taxa H2 and H4 of the unknown <italic><named-content content-type="taxon-name">Lucanus</named-content></italic> sp. and <italic><named-content content-type="taxon-name">Lucanus cervus judaicus</named-content></italic> (K2P-distance of 0.018 and 0.016, respectively). The remaining distances between (sub)species ranged from 0.087 and 0.179.</p><table-wrap id="T2" orientation="portrait" position="float"><label>Table 2.</label><caption><p>Intra- and interspecific K2P-distances for the 670 bp COI gene of western Palaearctic <italic><named-content content-type="taxon-name">Lucanus</named-content></italic> (sub)species. NA: intraspecific K2P-distance cannot be presented because only one sample is available.</p></caption><table frame="hsides" rules="groups"><tbody><tr><th rowspan="1" colspan="1"></th><th rowspan="1" colspan="1"><italic><named-content content-type="taxon-name">Lucanus cervus cervus</named-content></italic></th><th rowspan="1" colspan="1"><italic><named-content content-type="taxon-name">Lucanus cervus pentaphyllus</named-content></italic></th><th rowspan="1" colspan="1"><italic><named-content content-type="taxon-name">Lucanus cervus turcicus</named-content></italic></th><th rowspan="1" colspan="1"><italic><named-content content-type="taxon-name">Lucanus cervus fabiani</named-content></italic></th><th rowspan="1" colspan="1"><italic><named-content content-type="taxon-name">Lucanus cervus akbesianus</named-content></italic></th><th rowspan="1" colspan="1"><italic><named-content content-type="taxon-name">Lucanus cervus judaicus</named-content></italic></th><th rowspan="1" colspan="1"><italic><named-content content-type="taxon-name">Lucanus cervus laticornis</named-content></italic></th><th rowspan="1" colspan="1"><italic><named-content content-type="taxon-name">Lucanus ibericus</named-content></italic></th><th rowspan="1" colspan="1"><italic><named-content content-type="taxon-name">Lucanus tetraodon</named-content></italic></th><th rowspan="1" colspan="1"><italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) macrophyllus</named-content></italic></th><th rowspan="1" colspan="1"><italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) barbarossa</named-content></italic></th><th rowspan="1" colspan="1">unknown <italic><named-content content-type="taxon-name">Lucanus</named-content></italic> sp.</th></tr><tr><td rowspan="1" colspan="1"><bold><italic><named-content content-type="taxon-name">Lucanus cervus cervus</named-content></italic></bold></td><td rowspan="1" colspan="1">0–0.018</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1"><bold><italic><named-content content-type="taxon-name">Lucanus cervus pentaphyllus</named-content></italic></bold></td><td rowspan="1" colspan="1">0–0.018</td><td rowspan="1" colspan="1">0–0.014</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1"><bold><italic><named-content content-type="taxon-name">Lucanus cervus turcicus</named-content></italic></bold></td><td rowspan="1" colspan="1">0.001–0.021</td><td rowspan="1" colspan="1">0.003–0.017</td><td rowspan="1" colspan="1">0–0.017</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1"><bold><italic><named-content content-type="taxon-name">Lucanus cervus fabiani</named-content></italic></bold></td><td rowspan="1" colspan="1">0.161–0.167</td><td rowspan="1" colspan="1">0.160–0.163</td><td rowspan="1" colspan="1">0.159–0.169</td><td rowspan="1" colspan="1">NA</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1"><bold><italic><named-content content-type="taxon-name">Lucanus cervus akbesianus</named-content></italic></bold></td><td rowspan="1" colspan="1">0.118–0.161</td><td rowspan="1" colspan="1">0.121–0.155</td><td rowspan="1" colspan="1">0.121–0.165</td><td rowspan="1" colspan="1">0.159–0.174</td><td rowspan="1" colspan="1">0–0.045</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1"><bold><italic><named-content content-type="taxon-name">Lucanus cervus judaicus</named-content></italic></bold></td><td rowspan="1" colspan="1">0.151–0.164</td><td rowspan="1" colspan="1">0.153–0.160</td><td rowspan="1" colspan="1">0.155–0.170</td><td rowspan="1" colspan="1">0.167</td><td rowspan="1" colspan="1">0.144–0.154</td><td rowspan="1" colspan="1">NA</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1"><bold><italic><named-content content-type="taxon-name">Lucanus cervus laticornis</named-content></italic></bold></td><td rowspan="1" colspan="1">0.134–0.160</td><td rowspan="1" colspan="1">0.134–0.155</td><td rowspan="1" colspan="1">0.132–0.164</td><td rowspan="1" colspan="1">0.162–0.165</td><td rowspan="1" colspan="1">0.135–0.150</td><td rowspan="1" colspan="1">0.089–0.094</td><td rowspan="1" colspan="1">0.085</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1"><bold><italic><named-content content-type="taxon-name">Lucanus ibericus</named-content></italic></bold></td><td rowspan="1" colspan="1">0.029–0.039</td><td rowspan="1" colspan="1">0.034–0.035</td><td rowspan="1" colspan="1">0.028–0.037</td><td rowspan="1" colspan="1">0.174</td><td rowspan="1" colspan="1">0.132–0.151</td><td rowspan="1" colspan="1">0.174</td><td rowspan="1" colspan="1">0.141–0.168</td><td rowspan="1" colspan="1">NA</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1"><bold><italic><named-content content-type="taxon-name">Lucanus tetraodon</named-content></italic></bold></td><td rowspan="1" colspan="1">0.125–0.129</td><td rowspan="1" colspan="1">0.124–0.128</td><td rowspan="1" colspan="1">0.122–0.130</td><td rowspan="1" colspan="1">0.168–0.179</td><td rowspan="1" colspan="1">0.098–0.123</td><td rowspan="1" colspan="1">0.151–0.156</td><td rowspan="1" colspan="1">0.132–0.151</td><td rowspan="1" colspan="1">0.131–0.136</td><td rowspan="1" colspan="1">0.024</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1"><bold><italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) macrophyllus</named-content></italic></bold></td><td rowspan="1" colspan="1">0–0.012</td><td rowspan="1" colspan="1">0–0.014</td><td rowspan="1" colspan="1">0.006–0.015</td><td rowspan="1" colspan="1">0.159</td><td rowspan="1" colspan="1">0.116–0.141</td><td rowspan="1" colspan="1">0.147</td><td rowspan="1" colspan="1">0.130–0.145</td><td rowspan="1" colspan="1">0.028</td><td rowspan="1" colspan="1">0.120–0.124</td><td rowspan="1" colspan="1">NA</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1"><bold><italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) barbarossa</named-content></italic></bold></td><td rowspan="1" colspan="1">0.153–0.163</td><td rowspan="1" colspan="1">0.155–0.161</td><td rowspan="1" colspan="1">0.155–0.167</td><td rowspan="1" colspan="1">0.004–0.058</td><td rowspan="1" colspan="1">0.127–0.171</td><td rowspan="1" colspan="1">0.153–0.165</td><td rowspan="1" colspan="1">0.146–0.167</td><td rowspan="1" colspan="1">0.166–0.172</td><td rowspan="1" colspan="1">0.159–0.177</td><td rowspan="1" colspan="1">0.149–0.157</td><td rowspan="1" colspan="1">0.053</td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1"><bold>unknown <italic><named-content content-type="taxon-name">Lucanus</named-content></italic> sp.</bold></td><td rowspan="1" colspan="1">0.091–0.162</td><td rowspan="1" colspan="1">0.093–0.159</td><td rowspan="1" colspan="1">0.95–0.168</td><td rowspan="1" colspan="1">0.143–0.172</td><td rowspan="1" colspan="1">0.119–0.150</td><td rowspan="1" colspan="1">0.016–0.066</td><td rowspan="1" colspan="1">0.088–0.113</td><td rowspan="1" colspan="1">0.109–0.169</td><td rowspan="1" colspan="1">0.120–0.152</td><td rowspan="1" colspan="1">0.087–0.147</td><td rowspan="1" colspan="1">0.136–0.170</td><td rowspan="1" colspan="1">0.002–0.054</td></tr></tbody></table></table-wrap><table-wrap id="T3" orientation="portrait" position="float"><label>Table 3.</label><caption><p>Nucleotide composition and K2P-distances at each codon position of the 670 bp COI region.</p></caption><table frame="hsides" rules="groups"><tbody><tr><th rowspan="2" colspan="1"></th><th rowspan="1" colspan="3">Codon position</th></tr><tr><th rowspan="1" colspan="1">1<sup>st</sup></th><th rowspan="1" colspan="1">2<sup>nd</sup></th><th rowspan="1" colspan="1">3<sup>rd</sup></th></tr><tr><td rowspan="1" colspan="1"><bold>% A</bold></td><td rowspan="1" colspan="1">31.4</td><td rowspan="1" colspan="1">18.9</td><td rowspan="1" colspan="1">38.2</td></tr><tr><td rowspan="1" colspan="1"><bold>% T</bold></td><td rowspan="1" colspan="1">26.6</td><td rowspan="1" colspan="1">42.5</td><td rowspan="1" colspan="1">36.6</td></tr><tr><td rowspan="1" colspan="1"><bold>% G</bold></td><td rowspan="1" colspan="1">25.6</td><td rowspan="1" colspan="1">16.2</td><td rowspan="1" colspan="1">4.9</td></tr><tr><td rowspan="1" colspan="1"><bold>% C</bold></td><td rowspan="1" colspan="1">16.4</td><td rowspan="1" colspan="1">22.4</td><td rowspan="1" colspan="1">20.4</td></tr><tr><td rowspan="1" colspan="1"><bold>K2P-distance</bold></td><td rowspan="1" colspan="1">0–0.107</td><td rowspan="1" colspan="1">0–0.032</td><td rowspan="1" colspan="1">0–0.999</td></tr></tbody></table></table-wrap><p>These results do not show a distinct barcoding gap or other threshold to distinguish putative species, which is chiefly due to a lack of phylogenetic resolution to differentiate the said species and subspecies. If we consider the taxa of the <italic><named-content content-type="taxon-name">Lucanus cervus cervus</named-content></italic> clade to be members of the same species, 99.4% of all intra(sub)specific comparisons showed K2P-distances below 5% and 99.8% of the pairwise inter(sub)specific distances were above 5%. Nucleotide diagnostics are listed in Appendix 2. No diagnostic combination of nucleotide positions and characters were found for the taxa of the <italic><named-content content-type="taxon-name">Lucanus cervus cervus</named-content></italic> clade, <italic><named-content content-type="taxon-name">Lucanus ibericus</named-content></italic> not included. As the number of species and the sample size per species are rather limited, the nucleotide diagnostics should be considered with caution.</p></sec></sec><sec sec-type="Discussion"><title>Discussion</title><p>The present study shows that the sequenced COI fragment could discriminate several of the investigated western Palaearctic <pmc-comment>PageBreak</pmc-comment><pmc-comment>PageBreak</pmc-comment><italic><named-content content-type="taxon-name">Lucanus</named-content></italic> species and alleged subspecies of <italic><named-content content-type="taxon-name">Lucanus cervus</named-content></italic>. Well differentiated species and subspecies were <italic><named-content content-type="taxon-name">Lucanus cervus akbesianus</named-content></italic>, <italic><named-content content-type="taxon-name">Lucanus cervus laticornis</named-content></italic> and <italic><named-content content-type="taxon-name">Lucanus tetraodon</named-content></italic>, as well as the two eastern Asian species <italic><named-content content-type="taxon-name">Lucanus formosanus</named-content></italic> and <italic><named-content content-type="taxon-name">Lucanus hermani</named-content></italic>. Difficulties in molecular identification remained between <italic><named-content content-type="taxon-name">Lucanus cervus fabiani</named-content></italic> and <italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) barbarossa</named-content></italic>, <italic><named-content content-type="taxon-name">Lucanus cervus judaicus</named-content></italic> and the unidentified <italic><named-content content-type="taxon-name">Lucanus</named-content></italic> species, and between taxa of the <italic><named-content content-type="taxon-name">Lucanus cervus cervus</named-content></italic> clade. Although thoroughly sampled within their distribution range, <italic><named-content content-type="taxon-name">Lucanus cervus cervus</named-content></italic> and <italic><named-content content-type="taxon-name">Lucanus cervus turcicus</named-content></italic> could not be discriminated with a barcoding approach. Likewise, three out of four samples of <italic><named-content content-type="taxon-name">Lucanus cervus pentaphyllus</named-content></italic> possessed the most common haplotype of <italic><named-content content-type="taxon-name">Lucanus cervus cervus</named-content></italic>. Next to introgression following recent or past hybridisation events, incomplete sorting of ancestral variation may be the reason for the polyphyletic pattern. It is not known if <italic><named-content content-type="taxon-name">Lucanus</named-content></italic> can be infected with the endosymbiotic bacteria <italic><named-content content-type="taxon-name">Wolbachia</named-content></italic>, which can cause mitochondrial introgression between closely related species (e.g. <xref ref-type="bibr" rid="B63">Whitworth et al. 2007</xref>). Nonetheless, infections with <italic><named-content content-type="taxon-name">Wolbachia</named-content></italic> are quite common among insects, and should be taken into account (<xref ref-type="bibr" rid="B27">Hilgenboecker et al. 2008</xref>). However, the shift from four to five or even six lamellar segments on the antennal club is, at least in this tree of maternal inheritance, not synapomorphic among all individuals, and the number of lamellae may represent a case of parallel evolution or a phenotypically plastic trait within <italic><named-content content-type="taxon-name">Lucanus cervus</named-content></italic>, such that <italic><named-content content-type="taxon-name">Lucanus pentaphyllus</named-content></italic> and <italic><named-content content-type="taxon-name">Lucanus turcicus</named-content></italic> may merely represent morphotypes of <italic><named-content content-type="taxon-name">Lucanus cervus</named-content></italic>. This hypothesis seems less likely for <italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) macrophyllus</named-content></italic>. Although this taxon’s haplotype only differed from the main <italic><named-content content-type="taxon-name">Lucanus cervus cervus</named-content></italic> haplotype, A3, by its seven ambiguous sites, it has a very distinct morphology. The same can be said about <italic><named-content content-type="taxon-name">Lucanus ibericus</named-content></italic>, which was part of the same clade, but showed higher pairwise K2P-distances (0.028–0.032) when comparing it to the other taxa of the clade. Lumping <italic><named-content content-type="taxon-name">Lucanus ibericus</named-content></italic> and <italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) macrophyllus</named-content></italic> together with the <italic><named-content content-type="taxon-name">Lucanus cervus</named-content></italic> subspecies <italic><named-content content-type="taxon-name">Lucanus cervus</named-content></italic>, <italic><named-content content-type="taxon-name">Lucanus turcicus</named-content></italic> and <italic><named-content content-type="taxon-name">Lucanus pentaphyllus</named-content></italic> seems therefore ill advice.</p><p>Like <pmc-comment>PageBreak</pmc-comment><italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) macrophyllus</named-content></italic>, one sample of <italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) barbarossa</named-content></italic>, SB6, was embedded in the <italic><named-content content-type="taxon-name">Lucanus cervus cervus</named-content></italic> clade, opposed to the other two samples that clustered with <italic><named-content content-type="taxon-name">Lucanus cervus fabiani</named-content></italic>. The taxa of the latter group showed K2P-distances between 0.004 and 0.058, which indicates a close relationship between <italic><named-content content-type="taxon-name">Lucanus cervus fabiani</named-content></italic> and <italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) barbarossa</named-content></italic>, as well as <italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) barbarossa</named-content></italic> being very variable. High intraspecific variability could be indicative of cryptic diversity or population structure (<named-content content-type="taxon-name">Diptera</named-content>: <xref ref-type="bibr" rid="B40">Meier et al. 2006</xref>; <named-content content-type="taxon-name">Lycaenidae</named-content>: <xref ref-type="bibr" rid="B64">Wiemers and Fiedler 2007</xref>; <named-content content-type="taxon-name">Coleoptera</named-content>, <named-content content-type="taxon-name">Nitidulidae</named-content>: <xref ref-type="bibr" rid="B10">De Biase et al. 2012</xref>; <named-content content-type="taxon-name">Hemiptera</named-content>, <named-content content-type="taxon-name">Cicadidae</named-content>: <xref ref-type="bibr" rid="B46">Nunes et al. 2013</xref>). Despite the moderate to low genetic distance between <italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) barbarossa</named-content></italic> and <italic><named-content content-type="taxon-name">Lucanus cervus fabiani</named-content></italic>, these taxa are morphologically very distinct. This leaves us with either incomplete lineage sorting or introgression. Considering that both taxa have very proximate distribution ranges, introgressive hybridisation is likely. Even complete loss of the original mitochondrial genome of a species, resulting in a species with only mitochondrial genomes of the introgressed species is not unheard of (<xref ref-type="bibr" rid="B20">Hailer et al. 2012</xref>). Likewise, as <italic><named-content content-type="taxon-name">Lucanus cervus cervus</named-content></italic> and <italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) barbarossa</named-content></italic> occur sympatrically in Spain and Portugal (<xref ref-type="bibr" rid="B41">Méndez 2003</xref>), recent hybridisation and introgression cannot be ruled out as another or supplementary cause of the polyphyletic status of <italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) barbarossa</named-content></italic> (<xref ref-type="bibr" rid="B2">Avise 2000</xref>). Because SB6 merely differed from A3 at its five ambiguous sites, it could be perceived as a shared haplotype, which would corroborate this hypothesis (e.g. <xref ref-type="bibr" rid="B44">Nicholls et al. 2012</xref>). <italic><named-content content-type="taxon-name">Lucanus cervus akbesianus</named-content></italic>, <italic><named-content content-type="taxon-name">Lucanus cervus laticornis</named-content></italic> and <italic><named-content content-type="taxon-name">Lucanus cervus judaicus</named-content></italic> also have overlapping distributions. The former two were even sampled on the same tree in a Turkish forest (M. A. Cimaz, personal communication). In captivity, they do not seem to interbreed, which is concordant with our reporting of no shared haplotypes.</p><p>Finally, the <italic><named-content content-type="taxon-name">Lucanus</named-content></italic> samples from Israel and Lebanon that were unidentified at the species level, seemed closely related and formed a paraphyletic clade with <italic><named-content content-type="taxon-name">Lucanus cervus judaicus</named-content></italic>. Nevertheless, some of these samples could well be of a different species, indicated by the higher pairwise genetic distances (0.042–0.066). A detailed morphological and phylogenetic study is required here to investigate the number of species and relationship with <italic><named-content content-type="taxon-name">Lucanus cervus judaicus</named-content></italic>.</p><p>A distinct barcoding gap was absent for several species and subspecies of <italic><named-content content-type="taxon-name">Lucanus</named-content></italic>. This may either represent a low phylogenetic signal from the COI fragment for some relationships, a problem of basing a taxonomy on just one or a few morphological traits, or both. The use of the COI gene for barcoding purposes has had mixed results. High intraspecific variability (<xref ref-type="bibr" rid="B11">DeSalle et al. 2005</xref>) and closely related species (e.g. <xref ref-type="bibr" rid="B17">Funk and Omland 2003</xref>, <xref ref-type="bibr" rid="B21">Hajibabaei et al. 2006</xref>) can lead to an overlap in genetic distances, making the technique ineffective, as was shown here. In addition, NUMTs may complicate results and could cause the number of species to be overestimated (<xref ref-type="bibr" rid="B56">Song et al. 2008</xref>). Besides, the evolutionary history of the gene in question could be different from that of the studied species (<xref ref-type="bibr" rid="B39">Maddison 1997</xref>, <xref ref-type="bibr" rid="B14">Edwards 2009</xref>). Consequently, other or additional genes, ribosomal or nuclear, are recommended for barcoding purposes (<xref ref-type="bibr" rid="B13">Dupuis et al. 2012</xref>).</p></sec><sec sec-type="Conclusions"><title>Conclusions</title><p>This study revealed that while the 3’ terminus of COI contained sufficient information to resolve relationships among a number of closely related taxa, many others could not be robustly discriminated. Genotyping of additional specimens, especially of <pmc-comment>PageBreak</pmc-comment><italic><named-content content-type="taxon-name">Lucanus (Pseudolucanus) macrophyllus</named-content></italic>, <italic><named-content content-type="taxon-name">Lucanus ibericus</named-content></italic>, <italic><named-content content-type="taxon-name">Lucanus cervus judaicus</named-content></italic>, <italic><named-content content-type="taxon-name">Lucanus cervus fabiani</named-content></italic> and <italic><named-content content-type="taxon-name">Lucanus cervus laticornis</named-content></italic>, as well as all western Palaearctic taxa is needed to fully explore COI genetic diversity and to investigate the roles of phenotypic plasticity, hybridisation and incomplete lineage sorting underlying stag beetle biodiversity and inform taxonomic investigations. We therefore see this study as a starting point for future research which should also endeavour to combine analysis of nuclear markers, such as the internal transcribed spacer (ITS) and 28S rRNA gene (e.g. <xref ref-type="bibr" rid="B55">Smith et al. 2007</xref>), in combination with a detailed morphological investigation, to find a useful molecular identification tool for all western Palaearctic <italic><named-content content-type="taxon-name">Lucanus</named-content></italic> sp.</p></sec><sec><title>Authors’ contributions</title><p>The work presented here was carried out in collaboration between all authors. AT, KDG, GA, PA and LB defined the subject and the design of the study. KDG designed methods and experiments in the laboratory and supervised laboratory work. KC analysed the data, interpreted results and wrote the paper. AT was responsible for collecting the samples and co-wrote the taxonomical part of the paper. JM discussed analyses. GA, ES, NMcK and PS provided five sequences and revised primarily the material and methods section and the interpretation of the results. MZ, LB and PA provided samples and co-wrote the paper, particularly the taxonomical section. DH and RM provided samples. All authors have contributed to, revised and approved the manuscript.</p></sec></body><back><ack><title>Acknowledgements</title><p>We want to thank the following people for generously providing tissue samples of stag beetles or information: E. Atay, M. Avci, L. Barbiero, R. Bekchiev, G. Bonamie, S. Boucher, C. Bouget, H. Brustel, D. G. Carrilero, L.R. Castro, G. De Coninck, J. Ibero Caballero, A.M. Cimaz, I. de las Monjas, M. Fremlin, N. Gouix, C. Hawes, J-P. Huang, N. Jansson, A. Kairouz, S. Korneyev, V.A. Korneyev, D. Kovalchuk, I. López Pérez, Á. Martínez García, M. Méndez, M. Murat, L. Nádai, I. Nel, E. Orbach, H. Podskalská, S. Rastrero Sánchez, S. Reicher, O. Rittner, F. Roviralta Peña, P. Šípek, L. Valladares, J.T. Smit, Á. R. Quirós Menéndez. Also many thanks to Leen Verschaeve, Nancy Van Liefferinge, An Van Breusegem, David Halfmaerten and Sabrina Neyrinck (INBO) for laboratory assistance. 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</ref-list><app-group><app id="APP1"><title>Appendix 1</title><supplementary-material content-type="local-data" id="S1"><caption><p>Consensus Bayesian tree of 60 haplotypes of the 3’ end of the COI gene. Values given by the nodes are posterior probabilities above 0.70. (doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.3897/zookeys.365.5526.app1">10.3897/zookeys.365.5526.app1</ext-link>) File format: Adobe PDF file (pdf).</p></caption><media xlink:href="ZooKeys-365-105-s001.pdf" mimetype="Adobe PDF file" mime-subtype="pdf" orientation="portrait" xlink:type="simple" id="d36e6187" position="anchor"></media></supplementary-material></app><app id="APP2"><title>Appendix 2</title><supplementary-material content-type="local-data" id="S2"><caption><p>Nucleotide diagnostics for (sub)species or species groups according to the Neighbour-Joining and Bayesian Inference tree topology. (doi: <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.3897/zookeys.365.5526.app2">10.3897/zookeys.365.5526.app2</ext-link>) File format: Adobe PDF file (pdf).</p></caption><media xlink:href="ZooKeys-365-105-s002.pdf" mimetype="Adobe PDF file" mime-subtype="pdf" orientation="portrait" xlink:type="simple" id="d36e6198" position="anchor"></media></supplementary-material></app></app-group></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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