DNA Barcoding of genus Hexacentrus in China reveals cryptic diversity within Hexacentrusjaponicus (Orthoptera, Tettigoniidae)
Identifieur interne : 000106 ( Pmc/Corpus ); précédent : 000105; suivant : 000107DNA Barcoding of genus Hexacentrus in China reveals cryptic diversity within Hexacentrusjaponicus (Orthoptera, Tettigoniidae)
Auteurs : Hui-Fang Guo ; Bei Guan ; Fu-Ming Shi ; Zhi-Jun ZhouSource :
- ZooKeys [ 1313-2989 ] ; 2016.
Abstract
DNA barcoding has been proved successful to provide resolution beyond the boundaries of morphological information. Hence, a study was undertaken to establish DNA barcodes for all morphologically determined
Url:
DOI: 10.3897/zookeys.596.8669
PubMed: 27408576
PubMed Central: 4926654
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">DNA Barcoding of genus <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic> in China reveals cryptic diversity within <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">japonicus</named-content></named-content></italic> (<named-content content-type="taxon-name"><named-content content-type="order">Orthoptera</named-content></named-content>, <named-content content-type="taxon-name"><named-content content-type="family">Tettigoniidae</named-content></named-content>)</title><author><name sortKey="Guo, Hui Fang" sort="Guo, Hui Fang" uniqKey="Guo H" first="Hui-Fang" last="Guo">Hui-Fang Guo</name><affiliation><nlm:aff id="A1"><addr-line>The Key Laboratory of Zoological Systematics and Application, College of Life Sciences, Hebei University, Baoding 071002, China</addr-line></nlm:aff></affiliation></author><author><name sortKey="Guan, Bei" sort="Guan, Bei" uniqKey="Guan B" first="Bei" last="Guan">Bei Guan</name><affiliation><nlm:aff id="A1"><addr-line>The Key Laboratory of Zoological Systematics and Application, College of Life Sciences, Hebei University, Baoding 071002, China</addr-line></nlm:aff></affiliation></author><author><name sortKey="Shi, Fu Ming" sort="Shi, Fu Ming" uniqKey="Shi F" first="Fu-Ming" last="Shi">Fu-Ming Shi</name><affiliation><nlm:aff id="A1"><addr-line>The Key Laboratory of Zoological Systematics and Application, College of Life Sciences, Hebei University, Baoding 071002, China</addr-line></nlm:aff></affiliation></author><author><name sortKey="Zhou, Zhi Jun" sort="Zhou, Zhi Jun" uniqKey="Zhou Z" first="Zhi-Jun" last="Zhou">Zhi-Jun Zhou</name><affiliation><nlm:aff id="A1"><addr-line>The Key Laboratory of Zoological Systematics and Application, College of Life Sciences, Hebei University, Baoding 071002, China</addr-line></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">27408576</idno><idno type="pmc">4926654</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4926654</idno><idno type="RBID">PMC:4926654</idno><idno type="doi">10.3897/zookeys.596.8669</idno><date when="2016">2016</date><idno type="wicri:Area/Pmc/Corpus">000106</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">DNA Barcoding of genus <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic> in China reveals cryptic diversity within <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">japonicus</named-content></named-content></italic> (<named-content content-type="taxon-name"><named-content content-type="order">Orthoptera</named-content></named-content>, <named-content content-type="taxon-name"><named-content content-type="family">Tettigoniidae</named-content></named-content>)</title><author><name sortKey="Guo, Hui Fang" sort="Guo, Hui Fang" uniqKey="Guo H" first="Hui-Fang" last="Guo">Hui-Fang Guo</name><affiliation><nlm:aff id="A1"><addr-line>The Key Laboratory of Zoological Systematics and Application, College of Life Sciences, Hebei University, Baoding 071002, China</addr-line></nlm:aff></affiliation></author><author><name sortKey="Guan, Bei" sort="Guan, Bei" uniqKey="Guan B" first="Bei" last="Guan">Bei Guan</name><affiliation><nlm:aff id="A1"><addr-line>The Key Laboratory of Zoological Systematics and Application, College of Life Sciences, Hebei University, Baoding 071002, China</addr-line></nlm:aff></affiliation></author><author><name sortKey="Shi, Fu Ming" sort="Shi, Fu Ming" uniqKey="Shi F" first="Fu-Ming" last="Shi">Fu-Ming Shi</name><affiliation><nlm:aff id="A1"><addr-line>The Key Laboratory of Zoological Systematics and Application, College of Life Sciences, Hebei University, Baoding 071002, China</addr-line></nlm:aff></affiliation></author><author><name sortKey="Zhou, Zhi Jun" sort="Zhou, Zhi Jun" uniqKey="Zhou Z" first="Zhi-Jun" last="Zhou">Zhi-Jun Zhou</name><affiliation><nlm:aff id="A1"><addr-line>The Key Laboratory of Zoological Systematics and Application, College of Life Sciences, Hebei University, Baoding 071002, China</addr-line></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="2016">2016</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><label>Abstract</label><p>DNA barcoding has been proved successful to provide resolution beyond the boundaries of morphological information. Hence, a study was undertaken to establish DNA barcodes for all morphologically determined <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic> species in China collections. In total, 83 specimens of five <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic> species were barcoded using standard mitochondrial cytochrome c oxidase subunit I (COI) gene. Except for <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">japonicus</named-content></named-content></italic>, barcode gaps were present in the remaining <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic> species. Taxon ID tree generated seven <abbrev xlink:title="Barcoding of Life Data systems">BOLD</abbrev>’s barcode index numbers (BINs), four of which were in agreement with the morphological species. For <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">japonicus</named-content></named-content></italic>, the maximum intraspecific divergence (4.43%) produced a minimal overlap (0.64%), and 19 specimens were divided into three different BINs. There may be cryptic species within the current <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">japonicus</named-content></named-content></italic>. This study adds to a growing body of DNA barcodes that have become available for katydids, and shows that a DNA barcoding approach enables the identification of known <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic> species with a very high resolution.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Bensasson, D" uniqKey="Bensasson D">D Bensasson</name></author><author><name sortKey="Zhang, Dx" uniqKey="Zhang D">DX Zhang</name></author><author><name sortKey="Hewitt, Gm" uniqKey="Hewitt G">GM Hewitt</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bey Bienko, Gy" uniqKey="Bey Bienko G">GY Bey-Bienko</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Burland, Tg" uniqKey="Burland T">TG Burland</name></author></analytic></biblStruct><biblStruct><analytic><author><name 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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">27408576</article-id><article-id pub-id-type="pmc">4926654</article-id><article-id pub-id-type="doi">10.3897/zookeys.596.8669</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group></article-categories><title-group><article-title>DNA Barcoding of genus <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic> in China reveals cryptic diversity within <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">japonicus</named-content></named-content></italic> (<named-content content-type="taxon-name"><named-content content-type="order">Orthoptera</named-content></named-content>, <named-content content-type="taxon-name"><named-content content-type="family">Tettigoniidae</named-content></named-content>)</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Guo</surname><given-names>Hui-Fang</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Guan</surname><given-names>Bei</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Shi</surname><given-names>Fu-Ming</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Zhou</surname><given-names>Zhi-Jun</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib></contrib-group><aff id="A1"><label>1</label><addr-line>The Key Laboratory of Zoological Systematics and Application, College of Life Sciences, Hebei University, Baoding 071002, China</addr-line></aff><author-notes><corresp>Corresponding author: Zhi-Jun Zhou (<email xlink:type="simple">zhijunzhou@163.com</email>)</corresp><fn fn-type="edited-by"><p>Academic editor: F. Montealegre-Z</p></fn></author-notes><pub-date pub-type="collection"><year>2016</year></pub-date><pub-date pub-type="epub"><day>7</day><month>6</month><year>2016</year></pub-date><issue>596</issue><fpage>53</fpage><lpage>63</lpage><history><date date-type="received"><day>31</day><month>3</month><year>2016</year></date><date date-type="accepted"><day>25</day><month>5</month><year>2016</year></date></history><permissions><copyright-statement>Hui-Fang Guo, Bei Guan, Fu-Ming Shi, Zhi-Jun Zhou</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><self-uri content-type="zoobank" xlink:type="simple" xlink:href="http://zoobank.org/C6C19258-1B3C-4CDC-B334-EA6F2C5B09A3">http://zoobank.org/C6C19258-1B3C-4CDC-B334-EA6F2C5B09A3</self-uri><abstract><label>Abstract</label><p>DNA barcoding has been proved successful to provide resolution beyond the boundaries of morphological information. Hence, a study was undertaken to establish DNA barcodes for all morphologically determined <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic> species in China collections. In total, 83 specimens of five <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic> species were barcoded using standard mitochondrial cytochrome c oxidase subunit I (COI) gene. Except for <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">japonicus</named-content></named-content></italic>, barcode gaps were present in the remaining <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic> species. Taxon ID tree generated seven <abbrev xlink:title="Barcoding of Life Data systems">BOLD</abbrev>’s barcode index numbers (BINs), four of which were in agreement with the morphological species. For <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">japonicus</named-content></named-content></italic>, the maximum intraspecific divergence (4.43%) produced a minimal overlap (0.64%), and 19 specimens were divided into three different BINs. There may be cryptic species within the current <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">japonicus</named-content></named-content></italic>. This study adds to a growing body of DNA barcodes that have become available for katydids, and shows that a DNA barcoding approach enables the identification of known <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic> species with a very high resolution.</p></abstract><kwd-group><label>Keywords</label><kwd>BOLD</kwd><kwd>China</kwd><kwd>DNA Barcoding</kwd><kwd><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></kwd><kwd>species delineation</kwd><pmc-comment>PageBreak</pmc-comment>
</kwd-group></article-meta><notes><sec sec-type="Citation"><title>Citation</title><p>Guo H-F, Guan B, Shi F-M, Zhou Z-J (2016) DNA Barcoding of genus <italic>Hexacentrus</italic> in China reveals cryptic diversity within <italic>Hexacentrus japonicus</italic> (Orthoptera, Tettigoniidae). ZooKeys 596: 53–63. doi: <ext-link ext-link-type="doi" xlink:href="10.3897/zookeys.596.8669">10.3897/zookeys.596.8669</ext-link></p></sec></notes></front><body><sec sec-type="Introduction"><title>Introduction</title><p>DNA barcoding employs short, standardized gene regions (5' segment of mitochondrial cytochrome oxidase subunit I for animals) as an internal tag to enable metazoan species identification (<xref rid="B13" ref-type="bibr">Hebert et al. 2003</xref>). <xref rid="B24" ref-type="bibr">Schmidt et al. (2015)</xref> found that DNA barcoding largely supported 250 years of classical taxonomy for central European bees. Unlike distinct species, closely related species offer a great challenge for phylogeny reconstruction and species identification with DNA barcoding due to overlapping genetic variation (<xref rid="B5" ref-type="bibr">Dai et al. 2012</xref>). For example, <xref rid="B27" ref-type="bibr">Versteirt et al. (2015)</xref> found that DNA barcoding offered a reliable framework for mosquito species identification in Belgium except for some closely related species. <xref rid="B32" ref-type="bibr">Zhou et al. (2012)</xref> found that molecular identification with DNA barcoding supported most traditional morphological species of genus <italic><named-content content-type="taxon-name"><named-content content-type="genus">Ruspolia</named-content></named-content></italic> in China.</p><p>In this study, our objective is to assess the utility of DNA barcoding for closely related katydid species, belonging to the genus <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic> (Serville, 1831) in China. <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic> is mainly distributed in Australian, Afrotropical and Oriental realms. <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic> is a particularly speciose genus, containing 24 known species (<xref rid="B8" ref-type="bibr">Eades et al. 2016</xref>). <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic> was the single genus within <named-content content-type="taxon-name"><named-content content-type="subfamily">Hexacentrinae</named-content></named-content>, which has been reported in China according to “<named-content content-type="taxon-name"><named-content content-type="order">Orthoptera</named-content></named-content> Species File” (<xref rid="B8" ref-type="bibr">Eades et al. 2016</xref>). Up to now, a total of six <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic> species have been reported, including <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">japonicus</named-content></named-content></italic> (Karny, 1907), <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">unicolor</named-content></named-content></italic> (Serville, 1831), <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">yunnaneus</named-content></named-content></italic> (Bey-Bienko, 1962), <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">fuscipes</named-content></named-content></italic> (Matsumura & Shiraki, 1908), <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">mundus</named-content></named-content></italic> (Walker, 1869) and <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">expansus</named-content></named-content></italic> (<xref rid="B30" ref-type="bibr">Wang and Shi 2005</xref>). Due to the rather difficult morphological discrimination between <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic> species, an interspecific molecular delineation was needed. To make <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic> more accessible to the scientific community, the open access project “BHC” had been initiated in the <abbrev><def><p>Barcoding of Life Data systems </p></def>(BOLD)</abbrev> (<xref rid="B20" ref-type="bibr">Ratnasingham and Hebert 2007</xref>). There are a few DNA barcoding study concentrated on <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic>, and the number of barcode sequences was limited in <abbrev>BOLD</abbrev>. The goals of this study are as following: (i) it will allow scientists with molecular capability but insufficient knowledge of <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic> taxonomy and systematics to recognize species and document the biodiversity of <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic>. (ii) For <named-content content-type="taxon-name"><named-content content-type="family">Tettigoniidae</named-content></named-content> taxonomists, it contributes to integrative taxonomic approaches, such as the elucidation of related species and clarification of problematic species groups, association of the sexes within one species, and the identification of new species (<xref rid="B11" ref-type="bibr">Gibbs 2009</xref>, <xref rid="B12" ref-type="bibr">2011</xref>, <xref rid="B18" ref-type="bibr">Packer et al. 2009</xref>, <xref rid="B24" ref-type="bibr">Schmidt et al. 2015</xref>). To this end, we checked for the presence of species barcode gaps and cryptic diversity within species. <abbrev><def><p>BOLD’s barcode index number </p></def>(BIN)</abbrev> analysis tool (<xref rid="B22" ref-type="bibr">Ratnasingham and Hebert 2013</xref>) was used to analyze <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic>.</p></sec><sec sec-type="materials|methods"><title>Material and methods</title><sec sec-type="Collection of specimens"><title>Collection of specimens</title><p>All specimens were collected by hand or sweeping method during their active season (July–November). <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic> species were all gathered in China from 12 localities, with <pmc-comment>PageBreak</pmc-comment>the latitude from 18.70°N to 41.80°N and the longitude from 97.83°E to 123.38°E. One or more specimens were chosen from each locality in order to include as many morphologically distinguishable individuals per site as possible. Specimens were collected and stored in 100% ethanol at -20 °C and were deposited in the Hebei University Museum. Species-level identification was based on the original morphological descriptions, locality data and additional information. Details on all specimens (sampling location, GPS coordinates, voucher number, <abbrev>BOLD</abbrev> number, etc.) are available within the “DNA Barcoding of <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic> in China, BHC” project in the Barcode of Life Data Systems (<abbrev>BOLD</abbrev>. <ext-link ext-link-type="uri" xlink:href="http://www.barcodinglife.org">www.barcodinglife.org</ext-link>).</p></sec><sec sec-type="DNA extraction, amplification and sequencing"><title>DNA extraction, amplification and sequencing</title><p>Total DNA was extracted from the muscle of one hind leg of each specimens using TIANamp Genomic DNA Kit in accordance with the manufacturer’s instructions. The standardized gene regions of animals DNA barcoding was amplified using the primers COBU (5'-TYT CAA CAA AYC AYA ARG ATA TTG G-3') and COBL (5'-TAA ACT TCW GGR TGW CCA AAR AAT CA-3') (<xref rid="B19" ref-type="bibr">Pan et al. 2006</xref>). The 50 μL <abbrev><def><p>polymerase chain reaction </p></def>(PCR)</abbrev> mixture contained 3 μL of template DNA, 5 μL of 10 × buffer, 4 μL of dNTP mix, 5 μL of each primer (10 μM each), 0.5 μL of <italic>Taq</italic> polymerase (5 U/μL), and 27.5 μL of water. The thermal profile was: 94 °C for 3 min, 34 cycles at 94 °C for 30 s, 49 °C for 30 s, and 72 °C for 90 s, and final extension at 72 °C for 8 min. <abbrev xlink:title="polymerase chain reaction">PCR</abbrev> products were visualized in 1% agarose gels electrophoresis. <abbrev xlink:title="polymerase chain reaction">PCR</abbrev> products were sequenced directly using ABI BigDye Terminator chemistry on ABI3730 automated sequencer (Applied Biosystems) in Genewiz Inc. (Beijing, China), and in both directions to minimize <abbrev xlink:title="polymerase chain reaction">PCR</abbrev> artifacts, ambiguities and base calling error.</p></sec><sec sec-type="Data analysis"><title>Data analysis</title><p>Consensus sequence of both directions was assembled using SeqMan in Lasergene and verification of ambiguities and unexpected stop codons were performed in EditSeq (<xref rid="B3" ref-type="bibr">Burland 2000</xref>). Sequence alignments were conducted using Clustal X 1.81 (<xref rid="B26" ref-type="bibr">Thompson et al. 1997</xref>) with default parameters. The both ends of the sequences matching the primer sequences were excised to remove artificial nucleotide similarity introduced by <abbrev xlink:title="polymerase chain reaction">PCR</abbrev> amplification, resulting in the final data sets for barcoding analysis.</p><p>The analyses were restricted to the subset of sequences, which met barcode standards (sequence length > 500bp, < 1% ambiguous bases, bidirectional sequencing, country specification). Intra- and inter-specific genetic distances were based on the <abbrev><def><p>Kimura-2-parameter </p></def>(K2P)</abbrev> model (Kimura 1980) using the ‘distance summary’ tool in <abbrev>BOLD</abbrev>. The barcode gap was defined by intraspecific vs. interspecific [<abbrev><def><p>nearest neighbor </p></def>(NN)</abbrev>] genetic distance of species. A globally unique identifier (i.e. <abbrev xlink:title="BOLD’s barcode index number">BIN</abbrev>) then was assigned to each sequence cluster, creating an interim taxonomic system because the <pmc-comment>PageBreak</pmc-comment>members of a particular <abbrev xlink:title="BOLD’s barcode index number">BIN</abbrev> often correspond to a biological species. Character based DNA barcoding used the nucleotide variation in each position across DNA regions as diagnostic characters.</p></sec></sec><sec sec-type="Results"><title>Results</title><p>COI sequences were recovered from 86 of the 91 specimens that were analyzed with barcode compliant records from 83 specimens representing five species. Three records have no barcode compliant records because of low quality of trace file. A number of 80 barcodes belong to four previously identified species whereas three analyzed specimens were only identified to genus level because they are female; they probably are <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">yunnaneus</named-content></named-content></italic> due to collection in Yunnan and separated from other specimens. The 658bp length sequences without indel (insertion/deletion) had full-length records. COI sequences were translated to amino acid sequences to check for stop codons and shifts in reading frame that might indicate the presence of nuclear mitochondrial copies (numts), but none were detected. Diagnostic character analysis was consistent with that of traditional external appearance discrimination. <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">expansus</named-content></named-content></italic> only having one specimen was not analyzed, thus lacking diagnostic character.</p><sec sec-type="Distance summary and Barcode Gap analysis"><title>Distance summary and Barcode Gap analysis</title><p>Mean intraspecies divergence was 1.32% (ranged between 0.57% and 2.43%), and maximum intraspecies divergence 4.43% was observed in <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">japonicus</named-content></named-content></italic> (Table <xref ref-type="table" rid="T1">1</xref>). When correcting for the uneven sample sizes of species, the within-species divergence decreased from 1.32% to 1.23%. Between <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic> species, the average <abbrev xlink:title="Kimura-2-parameter">K2P</abbrev> genetic distance was 12.54%, whereas minimum genetic distance only 3.79% (Table <xref ref-type="table" rid="T1">1</xref>).</p><table-wrap id="T1" orientation="portrait" position="float"><label>Table 1.</label><caption><p>Mean and maximum intraspecific and <abbrev><def><p>nearest neighbor </p></def>(NN)</abbrev> distance for all specimens.</p></caption><table frame="hsides" rules="all" id="TID0E56AE"><tbody><tr><th rowspan="1" colspan="1">Species</th><th rowspan="1" colspan="1">Mean intraspecific distance</th><th rowspan="1" colspan="1">Max intraspecific distance</th><th rowspan="1" colspan="1">Nearest neighbor</th><th rowspan="1" colspan="1">Distance to <abbrev xlink:title="nearest neighbor">NN</abbrev></th></tr><tr><td rowspan="1" colspan="1"><italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">expansus</named-content></named-content></italic></td><td rowspan="1" colspan="1">N/A</td><td rowspan="1" colspan="1">N/A</td><td rowspan="1" colspan="1"><italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">unicolor</named-content></named-content></italic></td><td rowspan="1" colspan="1">13.19</td></tr><tr><td rowspan="1" colspan="1"><italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">japonicus</named-content></named-content></italic></td><td rowspan="1" colspan="1">2.43</td><td rowspan="1" colspan="1">4.43</td><td rowspan="1" colspan="1"><italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">mundus</named-content></named-content></italic></td><td rowspan="1" colspan="1">3.79</td></tr><tr><td rowspan="1" colspan="1"><italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">mundus</named-content></named-content></italic></td><td rowspan="1" colspan="1">0.57</td><td rowspan="1" colspan="1">0.93</td><td rowspan="1" colspan="1"><italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">japonicus</named-content></named-content></italic></td><td rowspan="1" colspan="1">3.79</td></tr><tr><td rowspan="1" colspan="1"><italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic> sp.</td><td rowspan="1" colspan="1">0.72</td><td rowspan="1" colspan="1">1.08</td><td rowspan="1" colspan="1"><italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">unicolor</named-content></named-content></italic></td><td rowspan="1" colspan="1">9.72</td></tr><tr><td rowspan="1" colspan="1"><italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">unicolor</named-content></named-content></italic></td><td rowspan="1" colspan="1">1.19</td><td rowspan="1" colspan="1">3.79</td><td rowspan="1" colspan="1"><italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic> sp.</td><td rowspan="1" colspan="1">9.72</td></tr></tbody></table></table-wrap><p>Singleton species (<italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">expansus</named-content></named-content></italic>) were excluded from barcode gap analysis. Except for <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">japonicus</named-content></named-content></italic>, barcode gap was present in the remaining <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic> species (Fig. <xref ref-type="fig" rid="F1">1</xref>). Although the maximum intraspecific divergence of <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">unicolor</named-content></named-content></italic> was more than 2% (ranged between 0 and 3.79%), but still less than minimum interspecific between <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">unicolor</named-content></named-content></italic> and its <abbrev xlink:title="nearest neighbor">NN</abbrev> (Nearest Neighbor) <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic> spp. However, the maximum intraspecific divergence of <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">japonicus</named-content></named-content></italic> (4.43%) produced a minimal overlap (0.64%).</p><fig id="F1" orientation="portrait" position="float"><label>Figure 1.</label><caption><p>Barcode gap plot showed the distance to the <abbrev><def><p>nearest neighbor </p></def>(NN)</abbrev> vs. the maximum intraspecific distance <abbrev><def><p>Kimura-2-parameter </p></def>(K2P)</abbrev> for 83 specimens. Dots above the 1:1 line indicated the presence of a barcode gap.</p></caption><graphic id="oo_91641.jpg" xlink:href="zookeys-596-053-g001"></graphic></fig><pmc-comment>PageBreak</pmc-comment>
</sec><sec sec-type="Taxon ID tree analysis"><title>Taxon ID tree analysis</title><p>The taxon ID tree was divided into seven clades represented by different BINs (Fig. <xref ref-type="fig" rid="F2">2</xref>). <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">unicolor</named-content></named-content></italic>, <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">mundus</named-content></named-content></italic> and <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">expansus</named-content></named-content></italic> were composed well-supported monophyletic groups, which were fully congruent with the morphological species. <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">japonicus</named-content></named-content></italic> were divided into three different BINs. Although no morphological differences were observed among these three BINs, there might be cryptic species within the current <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">japonicus</named-content></named-content></italic>.</p><fig id="F2" orientation="portrait" position="float"><label>Figure 2.</label><caption><p>Taxon ID tree revealed seven well-differentiated haplogroups. Process ID, location, and BINs were shown in the tree. The clusters with a blue box indicated there may be two new putative ‘cryptic species’ within <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">japonicus</named-content></named-content></italic>. The clade with a black box indicated the specimen had more mutation within <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">unicolor</named-content></named-content></italic>.</p></caption><graphic id="oo_91642.jpg" xlink:href="zookeys-596-053-g002"></graphic></fig></sec><sec sec-type="Diagnostic characters analysis"><title>Diagnostic characters analysis</title><p>Forty-four diagnostic characters were found in the study (Table <xref ref-type="table" rid="T2">2</xref>). Four <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic> species had diagnostic characters and the success rate was 80%. The number of diagnostic character sites of <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic> spp. was no less than 20 (Table <xref ref-type="table" rid="T2">2</xref>), which may be caused by scarce specimens and by the relative distance of the phylogenetic relationship to others.</p><table-wrap id="T2" orientation="portrait" position="float"><label>Table 2.</label><caption><p>Character-based DNA barcodes for four <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic> species of COI gene. A_7* means A is at the 7th position.</p></caption><table frame="hsides" rules="all" id="TID0E2HAG"><tbody><tr><th rowspan="1" colspan="1">Species</th><th rowspan="1" colspan="1">Diagnostic characters</th><th rowspan="1" colspan="1">Characters no.</th><th rowspan="1" colspan="1">Specimen no.</th></tr><tr><td rowspan="1" colspan="1"><italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic> sp.</td><td rowspan="1" colspan="1">A_7* G_184 C_247 A_301 A_304 T_346 G_391 C_400 G_424 C_463 C_472 C_500 T_517 G_550 G_586 C_607 A_619 C_622 C_625 G_628</td><td rowspan="1" colspan="1">20</td><td rowspan="1" colspan="1">3</td></tr><tr><td rowspan="1" colspan="1"><italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">unicolor</named-content></named-content></italic></td><td rowspan="1" colspan="1">T_25 T_136 T_223 A_227 T_322 T_379 T_424 G_487 A_502 C_517 T_529 C_530 G_532 C_550 C_586 G_619 T_631</td><td rowspan="1" colspan="1">17</td><td rowspan="1" colspan="1">54</td></tr><tr><td rowspan="1" colspan="1"><italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">mundus</named-content></named-content></italic></td><td rowspan="1" colspan="1">T_34 T_118 C_187 T_397 A_643</td><td rowspan="1" colspan="1">5</td><td rowspan="1" colspan="1">6</td></tr><tr><td rowspan="1" colspan="1"><italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">japonicus</named-content></named-content></italic></td><td rowspan="1" colspan="1">T_460 C_514</td><td rowspan="1" colspan="1">2</td><td rowspan="1" colspan="1">19</td></tr></tbody></table></table-wrap><pmc-comment>PageBreak</pmc-comment>
<pmc-comment>PageBreak</pmc-comment>
</sec></sec><sec sec-type="Discussion"><title>Discussion</title><p>The present study evaluated the efficacy of using DNA barcodes for the identification of <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic> in China and provided a group of sequences associated with the identified species. Using these DNA barcoding, not only can one delineate the boundaries between species, but also assign taxonomic status to unknown specimens from known species.</p><p><italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">unicolor</named-content></named-content></italic> was controversial, and <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">plantaris</named-content></named-content></italic> (Burmeister, 1838) and <italic><named-content content-type="taxon-name"><named-content content-type="genus">Tedla</named-content><named-content content-type="species">sellata</named-content></named-content></italic> (Walker, 1869) were considered as its synonyms. <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">unicolor</named-content></named-content></italic> is distributed in south of the Yangtze River. In this study, however, one specimen was collected from Henan. In fact, molecular data support <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">unicolor</named-content></named-content></italic> as a single group, with all specimens sharing one <abbrev xlink:title="BOLD’s barcode index number">BIN</abbrev> (<abbrev>BOLD</abbrev>: ACD 7247). The specimen with black box (Fig. <xref ref-type="fig" rid="F2">2</xref>) came from Guizhou, and had more mutations compared to the other <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">unicolor</named-content></named-content></italic> specimens. We cannot be completely certain that this phenomenon was due to geographic isolation rather than sequencing or calibrating errors. <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">japonicus</named-content></named-content></italic> was closely related to <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">unicolor</named-content></named-content></italic>, and they are widely distributed in the southwest, central and south areas of China. The tegmina of male <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">japonicas</named-content></named-content></italic> was short and broad (about 2.75–3.00 times), whereas <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">unicolor</named-content></named-content></italic> was long and narrow (about 2.95–3.30 times) as long as broad (<xref rid="B30" ref-type="bibr">Wang and Shi 2005</xref>). Nevertheless, the analyses revealed that <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">japonicus</named-content></named-content></italic> ranged to Hebei and Liaoning. Interestingly, <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">japonicus</named-content></named-content></italic> contained three BINs (<abbrev>BOLD</abbrev>: ACX 8110, <abbrev>BOLD</abbrev>: ACD 8277, <abbrev>BOLD</abbrev>: ACD 8278) with high intraspecific distance (> 2%). This group included 20 specimens, in which no morphological differences were found. Therefore it is necessary to clarify the status of this species complex as it may include more species than currently recognized. <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">mundus</named-content></named-content></italic> was only recorded in Guangxi and Yunnan. In this study, four specimens from Hainan were also identified as <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">mundus</named-content></named-content></italic> because only 2–3 larger teeth in the middle part of stridulatory file, however there are 6–7 large teeth in <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">japonicas</named-content></named-content></italic> and <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">unicolor</named-content></named-content></italic> (<xref rid="B30" ref-type="bibr">Wang and Shi 2005</xref>). Thus <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">mundus</named-content></named-content></italic>’s distribution was enlarged. All specimens of <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">mundus</named-content></named-content></italic> were assigned as 1 <abbrev xlink:title="BOLD’s barcode index number">BIN</abbrev>, which clearly confirm a consistency between molecular and morphological analyses. <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">expansus</named-content></named-content></italic>, due to the obviously inflated male tegmina, was easy to identify only by the morphological method. <abbrev xlink:title="BOLD’s barcode index number">BIN</abbrev> assignments also revealed that <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">expansus</named-content></named-content></italic> was a separated clade with only a single male available for analysis. The specimens from Yunnan almost certainly represent the ‘true’ <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">yunnaneus</named-content></named-content></italic> because the type loca<pmc-comment>PageBreak</pmc-comment>tion of this species (Hekou, Yunnan) was in close proximity. Hence, further specimens are needed to be analyzed, especially male material.</p><p>DNA barcoding, as one effective tool in insect taxonomy, had been already applied widely. It can rapidly acquire molecular data, simplifying species classification and identification. Yet, DNA barcodes has been argued to be unreliable for consistent species identification by many authors due to a number of drawbacks (<xref rid="B7" ref-type="bibr">DeSalle et al. 2005</xref>; <xref rid="B31" ref-type="bibr">Will et al. 2005</xref>; <xref rid="B23" ref-type="bibr">Rubinoff et al. 2006</xref>; <xref rid="B9" ref-type="bibr">Ebach 2011</xref>). Recent speciation, incomplete lineage sorting, interspecific hybridization and infection by endosymbiotic bacteria such as <italic><named-content content-type="taxon-name"><named-content content-type="genus">Wolbachia</named-content></named-content></italic> (<xref rid="B10" ref-type="bibr">Funk and Omland 2003</xref>) may all interfere with the performance of DNA barcoding in insects (<xref rid="B28" ref-type="bibr">Virgilio et al. 2010</xref>). In this context, most of the species can be amplified successfully; however, five specimens cannot be translated and three specimens only had one represented trace file, which cannot meet the DNA barcoding standards. The deep inspection of trace files indicated that most of these failures arose from co-amplification of the bacterial endosymbiont <italic><named-content content-type="taxon-name"><named-content content-type="genus">Wolbachia</named-content></named-content></italic>, which disturbed normal interpretation of trace file. It had been estimated that <italic><named-content content-type="taxon-name"><named-content content-type="genus">Wolbachia</named-content></named-content></italic> is present in two-thirds of all insect species (<xref rid="B14" ref-type="bibr">Hilgenboecker et al. 2008</xref>). There was no reason to doubt the absence of <italic><named-content content-type="taxon-name"><named-content content-type="genus">Wolbachia</named-content></named-content></italic> in <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic>. <xref rid="B33" ref-type="bibr">Zhou et al. (2014)</xref> found that the nuclear sequence of mitochondrial (numts) reported in <italic><named-content content-type="taxon-name"><named-content content-type="genus">Mecopoda</named-content><named-content content-type="species">niponensis</named-content></named-content></italic> may form a separate clade. The same case was reported in <italic><named-content content-type="taxon-name"><named-content content-type="genus">Podisma</named-content><named-content content-type="species">pedestris</named-content></named-content></italic> (<xref rid="B1" ref-type="bibr">Bensasson et al. 2000</xref>). But in this study, the clades in <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">japonicus</named-content></named-content></italic> were not caused by numts for all sequences analyzed without translation early termination, base indel, frameshift mutations. On the other hand, geographic isolation was also rejected, for only the specimens from Yunnan included three BINs in this group. The most probable reason was the existence of cryptic species compared to other <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic> species.</p><p>Species boundaries were hard to delimit only based on morphologyas, and analyses including additional sources of information such as molecular data, biogeography, behavior and ecology has been called integrative taxonomy which has been shown to be very useful (<xref rid="B6" ref-type="bibr">Dayrat 2005</xref>). We are convinced that DNA barcoding can promote the <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic> species identification. Our study showed that all known <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content></named-content></italic> species could be delimitating rapidly through DNA barcoding in China, except for <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">japonicus</named-content></named-content></italic>. <italic><named-content content-type="taxon-name"><named-content content-type="genus">Hexacentrus</named-content><named-content content-type="species">japonicus</named-content></named-content></italic> was problematic when using <abbrev>BOLD</abbrev> analysis. Finally, regardless of the promising results, the incorporation of nuclear genes is valuable for species delimitation and might strengthen the results, as they are independent of the maternal inherited mitochondrial genes.</p><p>The ideal situation would be that each species was represented by sequence from its type material, particularly the holotype. Type specimens were also dried specimens and DNA degraded at different level, so not only amplification was difficult, but also the damage of specimens can’t be neglected. Recently, <xref rid="B21" ref-type="bibr">Prosser et al. (2016)</xref> successfully obtained sequences from century-old type specimens using <abbrev><def><p>next-generation sequencing </p></def>(NGS)</abbrev>. We believed that DNA barcoding is useful in revealing cryptic biodiversity, potentially facilitating traditional taxonomy in future.</p><pmc-comment>PageBreak</pmc-comment>
</sec></body><back><ack><title>Acknowledgements</title><p>We are grateful to the following people for collecting specimens and providing valuable comments during the manuscript preparation: Qiong Song; Ji–Yuan Feng; Zhi–Lin Chen; Bao–Jie Du; Xun Bian. 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