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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">The Complete Chloroplast Genome Sequences of Five <italic>Epimedium</italic> Species: Lights into Phylogenetic and Taxonomic Analyses</title><author><name sortKey="Zhang, Yanjun" sort="Zhang, Yanjun" uniqKey="Zhang Y" first="Yanjun" last="Zhang">Yanjun Zhang</name><affiliation><nlm:aff id="aff1"><institution>Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences</institution><country>Wuhan, China</country></nlm:aff></affiliation></author><author><name sortKey="Du, Liuwen" sort="Du, Liuwen" uniqKey="Du L" first="Liuwen" last="Du">Liuwen Du</name><affiliation><nlm:aff id="aff1"><institution>Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences</institution><country>Wuhan, China</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><institution>College of Life Science, University of Chinese Academy of Sciences</institution><country>Beijing, China</country></nlm:aff></affiliation></author><author><name sortKey="Liu, Ao" sort="Liu, Ao" uniqKey="Liu A" first="Ao" last="Liu">Ao Liu</name><affiliation><nlm:aff id="aff1"><institution>Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences</institution><country>Wuhan, China</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><institution>College of Life Science, University of Chinese Academy of Sciences</institution><country>Beijing, China</country></nlm:aff></affiliation></author><author><name sortKey="Chen, Jianjun" sort="Chen, Jianjun" uniqKey="Chen J" first="Jianjun" last="Chen">Jianjun Chen</name><affiliation><nlm:aff id="aff1"><institution>Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences</institution><country>Wuhan, China</country></nlm:aff></affiliation></author><author><name sortKey="Wu, Li" sort="Wu, Li" uniqKey="Wu L" first="Li" last="Wu">Li Wu</name><affiliation><nlm:aff id="aff1"><institution>Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences</institution><country>Wuhan, China</country></nlm:aff></affiliation></author><author><name sortKey="Hu, Weiming" sort="Hu, Weiming" uniqKey="Hu W" first="Weiming" last="Hu">Weiming Hu</name><affiliation><nlm:aff id="aff1"><institution>Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences</institution><country>Wuhan, China</country></nlm:aff></affiliation></author><author><name sortKey="Zhang, Wei" sort="Zhang, Wei" uniqKey="Zhang W" first="Wei" last="Zhang">Wei Zhang</name><affiliation><nlm:aff id="aff3"><institution>College of Life Sciences, Xinyang Normal University</institution><country>Xinyang, China</country></nlm:aff></affiliation></author><author><name sortKey="Kim, Kyunghee" sort="Kim, Kyunghee" uniqKey="Kim K" first="Kyunghee" last="Kim">Kyunghee Kim</name><affiliation><nlm:aff id="aff4"><institution>Department of Plant Science, College of Agriculture and Life Sciences, Plant Genomics and Breeding Institute, and Research Institute of Agriculture and Life Sciences, Seoul National University</institution><country>Seoul, South Korea</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff5"><institution>Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement, Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences</institution><country>Guangzhou, China</country></nlm:aff></affiliation></author><author><name sortKey="Lee, Sang Choon" sort="Lee, Sang Choon" uniqKey="Lee S" first="Sang-Choon" last="Lee">Sang-Choon Lee</name><affiliation><nlm:aff id="aff4"><institution>Department of Plant Science, College of Agriculture and Life Sciences, Plant Genomics and Breeding Institute, and Research Institute of Agriculture and Life Sciences, Seoul National University</institution><country>Seoul, South Korea</country></nlm:aff></affiliation></author><author><name sortKey="Yang, Tae Jin" sort="Yang, Tae Jin" uniqKey="Yang T" first="Tae-Jin" last="Yang">Tae-Jin Yang</name><affiliation><nlm:aff id="aff4"><institution>Department of Plant Science, College of Agriculture and Life Sciences, Plant Genomics and Breeding Institute, and Research Institute of Agriculture and Life Sciences, Seoul National University</institution><country>Seoul, South Korea</country></nlm:aff></affiliation></author><author><name sortKey="Wang, Ying" sort="Wang, Ying" uniqKey="Wang Y" first="Ying" last="Wang">Ying Wang</name><affiliation><nlm:aff id="aff5"><institution>Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement, Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences</institution><country>Guangzhou, China</country></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">27014326</idno><idno type="pmc">4791396</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4791396</idno><idno type="RBID">PMC:4791396</idno><idno type="doi">10.3389/fpls.2016.00306</idno><date when="2016">2016</date><idno type="wicri:Area/Pmc/Corpus">000313</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">The Complete Chloroplast Genome Sequences of Five <italic>Epimedium</italic> Species: Lights into Phylogenetic and Taxonomic Analyses</title><author><name sortKey="Zhang, Yanjun" sort="Zhang, Yanjun" uniqKey="Zhang Y" first="Yanjun" last="Zhang">Yanjun Zhang</name><affiliation><nlm:aff id="aff1"><institution>Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences</institution><country>Wuhan, China</country></nlm:aff></affiliation></author><author><name sortKey="Du, Liuwen" sort="Du, Liuwen" uniqKey="Du L" first="Liuwen" last="Du">Liuwen Du</name><affiliation><nlm:aff id="aff1"><institution>Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences</institution><country>Wuhan, China</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><institution>College of Life Science, University of Chinese Academy of Sciences</institution><country>Beijing, China</country></nlm:aff></affiliation></author><author><name sortKey="Liu, Ao" sort="Liu, Ao" uniqKey="Liu A" first="Ao" last="Liu">Ao Liu</name><affiliation><nlm:aff id="aff1"><institution>Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences</institution><country>Wuhan, China</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><institution>College of Life Science, University of Chinese Academy of Sciences</institution><country>Beijing, China</country></nlm:aff></affiliation></author><author><name sortKey="Chen, Jianjun" sort="Chen, Jianjun" uniqKey="Chen J" first="Jianjun" last="Chen">Jianjun Chen</name><affiliation><nlm:aff id="aff1"><institution>Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences</institution><country>Wuhan, China</country></nlm:aff></affiliation></author><author><name sortKey="Wu, Li" sort="Wu, Li" uniqKey="Wu L" first="Li" last="Wu">Li Wu</name><affiliation><nlm:aff id="aff1"><institution>Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences</institution><country>Wuhan, China</country></nlm:aff></affiliation></author><author><name sortKey="Hu, Weiming" sort="Hu, Weiming" uniqKey="Hu W" first="Weiming" last="Hu">Weiming Hu</name><affiliation><nlm:aff id="aff1"><institution>Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences</institution><country>Wuhan, China</country></nlm:aff></affiliation></author><author><name sortKey="Zhang, Wei" sort="Zhang, Wei" uniqKey="Zhang W" first="Wei" last="Zhang">Wei Zhang</name><affiliation><nlm:aff id="aff3"><institution>College of Life Sciences, Xinyang Normal University</institution><country>Xinyang, China</country></nlm:aff></affiliation></author><author><name sortKey="Kim, Kyunghee" sort="Kim, Kyunghee" uniqKey="Kim K" first="Kyunghee" last="Kim">Kyunghee Kim</name><affiliation><nlm:aff id="aff4"><institution>Department of Plant Science, College of Agriculture and Life Sciences, Plant Genomics and Breeding Institute, and Research Institute of Agriculture and Life Sciences, Seoul National University</institution><country>Seoul, South Korea</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff5"><institution>Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement, Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences</institution><country>Guangzhou, China</country></nlm:aff></affiliation></author><author><name sortKey="Lee, Sang Choon" sort="Lee, Sang Choon" uniqKey="Lee S" first="Sang-Choon" last="Lee">Sang-Choon Lee</name><affiliation><nlm:aff id="aff4"><institution>Department of Plant Science, College of Agriculture and Life Sciences, Plant Genomics and Breeding Institute, and Research Institute of Agriculture and Life Sciences, Seoul National University</institution><country>Seoul, South Korea</country></nlm:aff></affiliation></author><author><name sortKey="Yang, Tae Jin" sort="Yang, Tae Jin" uniqKey="Yang T" first="Tae-Jin" last="Yang">Tae-Jin Yang</name><affiliation><nlm:aff id="aff4"><institution>Department of Plant Science, College of Agriculture and Life Sciences, Plant Genomics and Breeding Institute, and Research Institute of Agriculture and Life Sciences, Seoul National University</institution><country>Seoul, South Korea</country></nlm:aff></affiliation></author><author><name sortKey="Wang, Ying" sort="Wang, Ying" uniqKey="Wang Y" first="Ying" last="Wang">Ying Wang</name><affiliation><nlm:aff id="aff5"><institution>Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement, Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences</institution><country>Guangzhou, China</country></nlm:aff></affiliation></author></analytic><series><title level="j">Frontiers in Plant Science</title><idno type="eISSN">1664-462X</idno><imprint><date when="2016">2016</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p><italic>Epimedium</italic> L. is a phylogenetically and economically important genus in the family Berberidaceae. We here sequenced the complete chloroplast (cp) genomes of four <italic>Epimedium</italic> species using Illumina sequencing technology via a combination of <italic>de novo</italic> and reference-guided assembly, which was also the first comprehensive cp genome analysis on <italic>Epimedium</italic> combining the cp genome sequence of <italic>E. koreanum</italic> previously reported. The five <italic>Epimedium</italic> cp genomes exhibited typical quadripartite and circular structure that was rather conserved in genomic structure and the synteny of gene order. However, these cp genomes presented obvious variations at the boundaries of the four regions because of the expansion and contraction of the inverted repeat (IR) region and the single-copy (SC) boundary regions. The <italic>trnQ</italic>-<italic>UUG</italic> duplication occurred in the five <italic>Epimedium</italic> cp genomes, which was not found in the other basal eudicotyledons. The rapidly evolving cp genome regions were detected among the five cp genomes, as well as the difference of simple sequence repeats (SSR) and repeat sequence were identified. Phylogenetic relationships among the five <italic>Epimedium</italic> species based on their cp genomes showed accordance with the updated system of the genus on the whole, but reminded that the evolutionary relationships and the divisions of the genus need further investigation applying more evidences. The availability of these cp genomes provided valuable genetic information for accurately identifying species, taxonomy and phylogenetic resolution and evolution of <italic>Epimedium</italic>, and assist in exploration and utilization of <italic>Epimedium</italic> plants.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Avent, T" uniqKey="Avent T">T. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Front Plant Sci</journal-id><journal-id journal-id-type="iso-abbrev">Front Plant Sci</journal-id><journal-id journal-id-type="publisher-id">Front. Plant Sci.</journal-id><journal-title-group><journal-title>Frontiers in Plant Science</journal-title></journal-title-group><issn pub-type="epub">1664-462X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">27014326</article-id><article-id pub-id-type="pmc">4791396</article-id><article-id pub-id-type="doi">10.3389/fpls.2016.00306</article-id><article-categories><subj-group subj-group-type="heading"><subject>Plant Science</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>The Complete Chloroplast Genome Sequences of Five <italic>Epimedium</italic> Species: Lights into Phylogenetic and Taxonomic Analyses</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Zhang</surname><given-names>Yanjun</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><uri xlink:type="simple" xlink:href="http://loop.frontiersin.org/people/309180/overview"></uri></contrib><contrib contrib-type="author"><name><surname>Du</surname><given-names>Liuwen</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Liu</surname><given-names>Ao</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><uri xlink:type="simple" xlink:href="http://loop.frontiersin.org/people/314032/overview"></uri></contrib><contrib contrib-type="author"><name><surname>Chen</surname><given-names>Jianjun</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Wu</surname><given-names>Li</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Hu</surname><given-names>Weiming</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><uri xlink:type="simple" xlink:href="http://loop.frontiersin.org/people/310968/overview"></uri></contrib><contrib contrib-type="author"><name><surname>Zhang</surname><given-names>Wei</given-names></name><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Kim</surname><given-names>Kyunghee</given-names></name><xref ref-type="aff" rid="aff4"><sup>4</sup></xref><xref ref-type="aff" rid="aff5"><sup>5</sup></xref></contrib><contrib contrib-type="author"><name><surname>Lee</surname><given-names>Sang-Choon</given-names></name><xref ref-type="aff" rid="aff4"><sup>4</sup></xref></contrib><contrib contrib-type="author"><name><surname>Yang</surname><given-names>Tae-Jin</given-names></name><xref ref-type="aff" rid="aff4"><sup>4</sup></xref><xref ref-type="author-notes" rid="fn001"><sup>*</sup></xref><uri xlink:type="simple" xlink:href="http://loop.frontiersin.org/people/326410/overview"></uri></contrib><contrib contrib-type="author"><name><surname>Wang</surname><given-names>Ying</given-names></name><xref ref-type="aff" rid="aff5"><sup>5</sup></xref><xref ref-type="author-notes" rid="fn002"><sup>*</sup></xref><uri xlink:type="simple" xlink:href="http://loop.frontiersin.org/people/155612/overview"></uri></contrib></contrib-group><aff id="aff1"><sup>1</sup><institution>Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences</institution><country>Wuhan, China</country></aff><aff id="aff2"><sup>2</sup><institution>College of Life Science, University of Chinese Academy of Sciences</institution><country>Beijing, China</country></aff><aff id="aff3"><sup>3</sup><institution>College of Life Sciences, Xinyang Normal University</institution><country>Xinyang, China</country></aff><aff id="aff4"><sup>4</sup><institution>Department of Plant Science, College of Agriculture and Life Sciences, Plant Genomics and Breeding Institute, and Research Institute of Agriculture and Life Sciences, Seoul National University</institution><country>Seoul, South Korea</country></aff><aff id="aff5"><sup>5</sup><institution>Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement, Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences</institution><country>Guangzhou, China</country></aff><author-notes><fn fn-type="edited-by"><p>Edited by: Daniel Pinero, Universidad Nacional Autónoma de México, Mexico</p></fn><fn fn-type="edited-by"><p>Reviewed by: Caiguo Zhang, University of Colorado, USA; Sithichoke Tangphatsornruang, National Center for Genetic Engineering and Biotechnology, Thailand</p></fn><corresp id="fn001">*Correspondence: Tae-Jin Yang <email xlink:type="simple">tjyang@snu.ac.kr</email>;</corresp><corresp id="fn002">Ying Wang <email xlink:type="simple">yingwang@scib.ac.cn</email></corresp><fn fn-type="other" id="fn003"><p>This article was submitted to Plant Genetics and Genomics, a section of the journal Frontiers in Plant Science</p></fn></author-notes><pub-date pub-type="epub"><day>15</day><month>3</month><year>2016</year></pub-date><pub-date pub-type="collection"><year>2016</year></pub-date><volume>7</volume><elocation-id>306</elocation-id><history><date date-type="received"><day>17</day><month>1</month><year>2016</year></date><date date-type="accepted"><day>26</day><month>2</month><year>2016</year></date></history><permissions><copyright-statement>Copyright © 2016 Zhang, Du, Liu, Chen, Wu, Hu, Zhang, Kim, Lee, Yang and Wang.</copyright-statement><copyright-year>2016</copyright-year><copyright-holder>Zhang, Du, Liu, Chen, Wu, Hu, Zhang, Kim, Lee, Yang and Wang</copyright-holder><license 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). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p><italic>Epimedium</italic> L. is a phylogenetically and economically important genus in the family Berberidaceae. We here sequenced the complete chloroplast (cp) genomes of four <italic>Epimedium</italic> species using Illumina sequencing technology via a combination of <italic>de novo</italic> and reference-guided assembly, which was also the first comprehensive cp genome analysis on <italic>Epimedium</italic> combining the cp genome sequence of <italic>E. koreanum</italic> previously reported. The five <italic>Epimedium</italic> cp genomes exhibited typical quadripartite and circular structure that was rather conserved in genomic structure and the synteny of gene order. However, these cp genomes presented obvious variations at the boundaries of the four regions because of the expansion and contraction of the inverted repeat (IR) region and the single-copy (SC) boundary regions. The <italic>trnQ</italic>-<italic>UUG</italic> duplication occurred in the five <italic>Epimedium</italic> cp genomes, which was not found in the other basal eudicotyledons. The rapidly evolving cp genome regions were detected among the five cp genomes, as well as the difference of simple sequence repeats (SSR) and repeat sequence were identified. Phylogenetic relationships among the five <italic>Epimedium</italic> species based on their cp genomes showed accordance with the updated system of the genus on the whole, but reminded that the evolutionary relationships and the divisions of the genus need further investigation applying more evidences. The availability of these cp genomes provided valuable genetic information for accurately identifying species, taxonomy and phylogenetic resolution and evolution of <italic>Epimedium</italic>, and assist in exploration and utilization of <italic>Epimedium</italic> plants.</p></abstract><kwd-group><kwd><italic>Epimedium</italic></kwd><kwd>chloroplast genome</kwd><kwd>genome structure</kwd><kwd>phylogenetic relationships</kwd><kwd>taxonomic identification</kwd></kwd-group><counts><fig-count count="6"></fig-count><table-count count="3"></table-count><equation-count count="0"></equation-count><ref-count count="53"></ref-count><page-count count="12"></page-count><word-count count="6721"></word-count></counts></article-meta></front><body><sec sec-type="intro" id="s1"><title>Introduction</title><p><italic>Epimedium</italic> comprising about 58 species, is a phylogenetically and economically important genus in the family Berberidaceae (Stearn, <xref rid="B39" ref-type="bibr">2002</xref>; Ying et al., <xref rid="B50" ref-type="bibr">2011</xref>). As the diversity center of <italic>Epimedium</italic>, China possesses about 48 species, and has used <italic>Epimedium</italic> plants as herb-medicine for more than 2000 years. Herb epimedii has been verified with activity in nourishing the kidney, reinforcing the Yang, regulating bone remodeling, curing cardiovascular diseases, possessing anti-cancer, and anti-aging benefits (Ma et al., <xref rid="B27" ref-type="bibr">2011</xref>; Jiang et al., <xref rid="B11" ref-type="bibr">2015</xref>). The kind and quantity of drugs and health products with herb epimedii as raw materials have been increasing in the last 20 years, which has led to substantial appreciation of prices of the medicinal materials. Furthermore, bearing attractive foliage and flowers, <italic>Epimedium</italic> plants were previously mainly introduced as perennial garden plant in Europe and America. At present, the horticultural values of <italic>Epimedium</italic> plants have been widely paid attention with great commercial prospects (Lubell and Brand, <xref rid="B26" ref-type="bibr">2005</xref>; Ren et al., <xref rid="B34" ref-type="bibr">2008</xref>; Avent, <xref rid="B1" ref-type="bibr">2010</xref>).</p><p><italic>Epimedium</italic> is taxonomically and phylogenetically regarded as one of the most challengingly difficult taxa in plants. The updated system of <italic>Epimedium</italic> classified the genus into two subgenera, four sections, and four series mainly based on geographical distribution, and leaf, and flower morphology (Stearn, <xref rid="B39" ref-type="bibr">2002</xref>). However, molecular phylogenetic analyses based on internal transcribed spacer (ITS), <italic>trnK</italic>-<italic>matK, atpB</italic>-<italic>rbcL</italic> spacer sequences, and amplified fragment length polymorphisms (AFLPs) only consistently supported subg. <italic>Rhizophyllum</italic> and four sections of subg. <italic>Epimedium</italic> as five distinctive clades (Sun et al., <xref rid="B40" ref-type="bibr">2005</xref>; Zhang et al., <xref rid="B51" ref-type="bibr">2007</xref>, <xref rid="B52" ref-type="bibr">2014</xref>; De Smet et al., <xref rid="B4" ref-type="bibr">2012</xref>). The two subgenera were not well-supported, the relationships between five clades were unresolved except for sect. <italic>Epimedium</italic> as sister to sect. <italic>Macroceras</italic>, as well as the four series of sect. <italic>Diphyllon</italic> being poorly supported. As a genus of basal eudicots in North Temperate Zone, the five clades of <italic>Epimedium</italic> have their unique distribution regions, respectively, and with enormous gaps. It needs more effective molecular markers to investigate the relationships between the five clades and classification system of <italic>Epimedium</italic>, as well as the origin, evolution, migration, and dispersal of the genus in North Temperate Zone.</p><p>It has been intractable for the species identification of <italic>Epimedium</italic>, particularly for those of sect. <italic>Diphyllon</italic>, which baffled the effective exploration and utilization of the genus. Chinese sect. <italic>Diphyllon</italic> has highest species diversity level with about 47 species, and sympatric distribution, and hybridization made the interspecies relationship very complicated. Furthermore, many species, such as <italic>E. sagittatum, E. pubescens</italic>, and <italic>E. acuminatum</italic>, have abundant infra-species variations in morphology and medicinal ingredients. However, only AFLPs were heretofore and successfully applied to identify the species of sect. <italic>Diphyllon</italic> (Zhang et al., <xref rid="B52" ref-type="bibr">2014</xref>). Internal primer binding sites (iPBS) were used to investigate the intra-species variations of <italic>E. sagittatum</italic> (Chen et al., <xref rid="B3" ref-type="bibr">2015</xref>). For conservation, utilization, and domestication of <italic>Epimedium</italic> plants, more effective molecular markers are needed to identify <italic>Epimedium</italic> species and conduct the population genetics and breeding for the <italic>Epimedium</italic> genus.</p><p>The chloroplast (cp) is an important plastid that plays a key role in plant cell for photosynthesis and carbon fixation (Neuhaus and Emes, <xref rid="B30" ref-type="bibr">2000</xref>). The cp genomes in angiosperms are circular DNA molecules ranging from 115 to 165 kb in length and consisting of two copies of a large inverted repeat (IR) region separated by a large-single-copy (LSC) region and a small-single-copy (SSC) region (Raubeson and Jansen, <xref rid="B33" ref-type="bibr">2005</xref>; Wicke et al., <xref rid="B46" ref-type="bibr">2011</xref>). The cp genomes could provide valuable information for taxonomy and phylogeny as a result of sequence divergence between plant species and individuals (Jansen et al., <xref rid="B10" ref-type="bibr">2007</xref>; Moore et al., <xref rid="B29" ref-type="bibr">2007</xref>; Parks et al., <xref rid="B31" ref-type="bibr">2009</xref>; Huang et al., <xref rid="B9" ref-type="bibr">2014</xref>; Jung et al., <xref rid="B12" ref-type="bibr">2014</xref>). Owing to being haploid, maternal inheritance, and high conservation in gene content and genome structure, the cp genomes have been popular to study the evolutionary relationships at almost any taxonomic level in plants. With the advent of high-throughput sequencing technologies, it is now more practical and inexpensive to obtain cp genome sequences and promote cp-based phylogenetics to phylogenomics.</p><p>In this study, we sequenced the cp genomes of four <italic>Epimedium</italic> species using the next-generation sequencing platform, which is also the first comprehensive analysis on cp genomes for <italic>Epimedium</italic> combining the cp genome of <italic>E. koreanum</italic> previously reported (Lee et al., <xref rid="B21" ref-type="bibr">2015</xref>). Our study aims were as follows: (1) to investigate global structural patterns of <italic>Epimedium</italic> cp genomes; (2) to screen sequence divergence hotspot regions in the five <italic>Epimedium</italic> cp genomes; (3) to examine variations of simple sequence repeats (SSRs) and repeat sequences among the five <italic>Epimedium</italic> cp genomes; (4) to reconstruct phylogenetic relationships among the five <italic>Epimedium</italic> species using their cp genome sequences. The results will provide abundant information for further species identification, taxonomy and phylogenetic resolution of <italic>Epimedium</italic>, and assist in exploration and utilization of <italic>Epimedium</italic> plants.</p></sec><sec sec-type="materials and methods" id="s2"><title>Materials and methods</title><sec><title>Sample preparation, sequencing, assembly, and validation</title><p>Fresh leaves of five <italic>Epimedium</italic> species, four from China, and one from Korea, were sampled. The samples of four Chinese species were used for complete cp genome sequencing, while that of <italic>E. koreanum</italic> from Korea was only used for PCR-based validating its cp genome sequence (KM207675) previously reported (Lee et al., <xref rid="B21" ref-type="bibr">2015</xref>). The voucher herbarium specimens of four Chinese species were deposited at the Herbaria of Wuhan Botanical Garden, Chinese Academy of Sciences (HIB), and the sample of <italic>E. koreanum</italic> was deposited at Wuhan Botanical Garden, Chinese Academy of Sciences, Hallym University and Seoul National University (Table <xref ref-type="supplementary-material" rid="SM1">S1</xref>). Total genomic DNA per species was extracted from 100 mg fresh leaves using the DNeasy Plant MiniKit (Qiagen, CA, USA).</p><p>For the four Chinese <italic>Epimedium</italic> species, Purified DNA (5 mg) was sheared by nebulization with compressed nitrogen gas, yielding fragments of 300 bp in length, and fragmentation quality was checked on a Bioanalyzer 2100 (Agilent Technologies). Paired-end libraries were constructed following the manufacturer's protocol (Illumina, San Diego, California, USA). Genomic DNAs of four species were sequenced on a single lane on HiSeq2000 flow cell lanes (Illumina Inc.) by National Instrumentation Center for Environmental Management (NICEM; <ext-link ext-link-type="uri" xlink:href="http://nature.snu.ac.kr/kr.php">http://nature.snu.ac.kr/kr.php</ext-link>), Seoul, Korea.</p><p>For each of the four Chinese <italic>Epimedium</italic> species, cp genome reads were extracted by mapping all raw reads to the reference cp genome of <italic>Nandina domestica</italic> (DQ923117) with BWA (Li and Durbin, <xref rid="B22" ref-type="bibr">2009</xref>). High quality reads were obtained using the CLC-quality trim tool with Phred scores of < 20 and assembled using the CLC genome assembler v4.06 (<ext-link ext-link-type="uri" xlink:href="http://www.clcbio.com/products/clc-assembly-cell">http://www.clcbio.com/products/clc-assembly-cell</ext-link>) with default parameters. Sequence gaps were filled by Gapcloser included in the SOAP package v1.12 (Li et al., <xref rid="B23" ref-type="bibr">2010</xref>). All the contigs were aligned to the reference cp genome of <italic>Nandina domestica</italic> using MUMmer (Kurtz et al., <xref rid="B19" ref-type="bibr">2004</xref>), and aligned contigs were ordered according to the reference cp genome. Based on the reference cp genome, the four junctions between LSC/IRs and SSC/IRs of the five sampled <italic>Epimedium</italic> species were validated with PCR-based conventional Sanger sequencing, respectively. To avoid assembly errors and obtain high quality complete cp genome sequences, validation of assembly was also carried out on 10 chloroplast genes (Table <xref ref-type="supplementary-material" rid="SM2">S2</xref>).</p></sec><sec><title>Genome annotation and analysis</title><p>Initial gene annotation of the five chloroplast genomes (including that of <italic>E. koreanum</italic>, KM207675) was performed with Dual Organellar GenoMe Annotator (DOGMA; Wyman et al., <xref rid="B47" ref-type="bibr">2004</xref>). DOGMA annotations were manually corrected for the start and stop codons and intron/exon boundaries by comparison to homologous genes from other sequenced cp genomes in Ranales. The tRNA genes were also verified with ARAGORN (Laslett and Canback, <xref rid="B20" ref-type="bibr">2004</xref>) and tRNAscan-SE (Lowe and Eddy, <xref rid="B25" ref-type="bibr">1997</xref>; Schattner et al., <xref rid="B37" ref-type="bibr">2005</xref>). The circular cp genome maps were drawn using the OrganellarGenome DRAW tool (ORDRAW; Lohse et al., <xref rid="B24" ref-type="bibr">2007</xref>), with subsequent manual editing.</p><p>Cp genome comparison among the five <italic>Epimedium</italic> species was performed with the mVISTA program (Frazer et al., <xref rid="B5" ref-type="bibr">2004</xref>). Genome, protein coding gene, intron, and spacer sequence divergences were evaluated using DnaSP 5.10 (Rozas et al., <xref rid="B35" ref-type="bibr">2003</xref>) after aligned. The genome sequences were aligned using MAFFT v5 (Katoh and Toh, <xref rid="B13" ref-type="bibr">2010</xref>) and adjusted manually where necessary. For the protein coding gene sequences, introns and spacers, every gene or fragment was edited using ClustalW multiple alignment option within the software BioEdit v7.0.9.0 (Hall, <xref rid="B7" ref-type="bibr">2011</xref>).</p><p>Microsatellites (mono-, di-, tri-, tetra-, penta-, and hexanucleotide repeats) were detected using the Perl script MISA (Thiel et al., <xref rid="B43" ref-type="bibr">2003</xref>) with thresholds of ten repeat units for mononucleotide SSRs, five repeat units for di- and trinucleotide SSRs, and three repeat units for tetra-, penta-, and hexanucleotide SSRs. Size and location of both direct (forward) and inverted (palindromic) repeats in the <italic>Epimedium</italic> cp genome were identified by running REPuter (Kurtz et al., <xref rid="B18" ref-type="bibr">2001</xref>) according to the following criteria: cutoff n ≥30% bp and 90% sequence identities (Hamming distance of 3).</p></sec><sec><title>Phylogenetic analysis</title><p>It was found that <italic>trnQ-UUG</italic> genes were duplicated in the LSC of the five <italic>Epimedium</italic> cp genomes, which was not found in other basal eudicotyledons. For investigating the evolution of <italic>trnQ-UUG</italic> gene of <italic>Epimedium</italic>, phylogenetic analyses was conducted based on the nucleotide sequence of the gene of <italic>Epimedium</italic> and other taxa of basal eudicots. The phylogenetic analyses were also performed for the five <italic>Epimedium</italic> species with <italic>Nandina domestica</italic> and <italic>Aconitum barbatum</italic> of Ranales as outgroups. The analyses were carried out based on the following three data sets: (1) the complete cp DNA sequences; (2) protein coding sequences; (3) the introns and spacers. The nucleotide sequence data of <italic>trnQ-UUG</italic> gene and cp genome, except those of the four Chinese <italic>Epimedium</italic> species, were obtained from NCBI, which the sequence data of <italic>trnQ-UUG</italic> gene were also obtained from the corresponding Genbank files of cp genome sequence data (Table <xref ref-type="supplementary-material" rid="SM3">S3</xref>).</p><p>Maximum parsimony (MP) analyses were conducted using PAUP v4b10 (Swofford, <xref rid="B41" ref-type="bibr">2003</xref>). Heuristic search were performed with 1000 random addition sequences, 10 trees held at each step, tree-bisection-reconnection (TBR) branch swapping and MulTrees switched off. Branch support was assessed with 1000 bootstrap replicates with 10 random taxon additions each and TBR and MulTtrees ON. Maximum likelihood (ML) analyses were performed using RAxML-HPC BlackBox v.8.1.24 on the CIPRES Science Gateway website (Stamatakis et al., <xref rid="B38" ref-type="bibr">2008</xref>; Miller et al., <xref rid="B28" ref-type="bibr">2010</xref>). The best-fitting model was selected using ModelTest v.0.1.1 (Posada, <xref rid="B32" ref-type="bibr">2008</xref>), and branch support was estimated with 1000 bootstrap replicates.</p></sec></sec><sec id="s3"><title>Results and discussions</title><sec><title>Genome assembly and PCR-Based validation</title><p>Using the Illumina HiSeq 2000 system, five <italic>Epimedium</italic> species were sequenced to produce 4,573,881–4,675,703 paired-end raw reads (101 bp in average reads length). After screening these paired-end reads through alignment with reference cp genomes of <italic>Nandina domestica</italic>, 84,589 to 236,730 cp genome reads were extracted with 50 × to 145 × coverage (Table <xref ref-type="table" rid="T1">1</xref>). Four junction regions and 10 cp genes were validated by PCR-based sequencing in each of the five <italic>Epimedium</italic> cp genomes. The PCR-based sequencing on <italic>E. koreanum</italic> demonstrated identical with its original <italic>de novo</italic> assembly of complete cp genome sequence (KM20267; Lee et al., <xref rid="B21" ref-type="bibr">2015</xref>). However, some initial gene annotations on the sequence were inaccurate, for example that only one <italic>trnQ-UUG</italic> was identified while two copies of <italic>trnQ-UUG</italic> were actually located in LSC. We hereon updated the annotation on the cp genome sequence of <italic>E. koreanum</italic> with Genbank accession number <ext-link ext-link-type="DDBJ/EMBL/GenBank" xlink:href="KU522471">KU522471</ext-link>. The four Chinese <italic>Epimedium</italic> cp genome sequences were also deposited in GenBank (accession numbers, <ext-link ext-link-type="DDBJ/EMBL/GenBank" xlink:href="KU522469">KU522469</ext-link>, <ext-link ext-link-type="DDBJ/EMBL/GenBank" xlink:href="KU522470">KU522470</ext-link>, <ext-link ext-link-type="DDBJ/EMBL/GenBank" xlink:href="KU522472">KU522472</ext-link>, and <ext-link ext-link-type="DDBJ/EMBL/GenBank" xlink:href="KU522473">KU522473</ext-link>).</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p><bold>Summary of the sequencing data for five <italic><bold>Epimedium</bold></italic> species</bold>.</p></caption><table frame="hsides" rules="groups"><thead><tr><th valign="top" align="left" rowspan="1" colspan="1"><bold>Species</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>Raw read no</bold>.</th><th valign="top" align="center" rowspan="1" colspan="1"><bold>Total read length (bp)</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>Mapped read no</bold>.</th><th valign="top" align="center" rowspan="1" colspan="1"><bold>Mapped to reference genome (%)</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>Cp genome coverage (×)</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>Cp genome length (bp)</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>LSC length (bp)</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>SSC length (bp)</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>IR length (bp)</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>GC content (%)</bold></th></tr></thead><tbody><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>E. acuminatum</italic></td><td valign="top" align="center" rowspan="1" colspan="1">4,638,760</td><td valign="top" align="center" rowspan="1" colspan="1">468,514,760</td><td valign="top" align="center" rowspan="1" colspan="1">84,589</td><td valign="top" align="center" rowspan="1" colspan="1">1.82</td><td valign="top" align="center" rowspan="1" colspan="1">49.78</td><td valign="top" align="center" rowspan="1" colspan="1">159,112</td><td valign="top" align="center" rowspan="1" colspan="1">86,561</td><td valign="top" align="center" rowspan="1" colspan="1">17,069</td><td valign="top" align="center" rowspan="1" colspan="1">27,741</td><td valign="top" align="center" rowspan="1" colspan="1">38.81</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>E. dolichostemon</italic></td><td valign="top" align="center" rowspan="1" colspan="1">4,622,454</td><td valign="top" align="center" rowspan="1" colspan="1">466,867,854</td><td valign="top" align="center" rowspan="1" colspan="1">138,527</td><td valign="top" align="center" rowspan="1" colspan="1">3.00</td><td valign="top" align="center" rowspan="1" colspan="1">84.83</td><td valign="top" align="center" rowspan="1" colspan="1">157,039</td><td valign="top" align="center" rowspan="1" colspan="1">88,394</td><td valign="top" align="center" rowspan="1" colspan="1">17,077</td><td valign="top" align="center" rowspan="1" colspan="1">25,784</td><td valign="top" align="center" rowspan="1" colspan="1">38.80</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>E. lishihchenii</italic></td><td valign="top" align="center" rowspan="1" colspan="1">4,675,703</td><td valign="top" align="center" rowspan="1" colspan="1">472,246,003</td><td valign="top" align="center" rowspan="1" colspan="1">99,587</td><td valign="top" align="center" rowspan="1" colspan="1">2.13</td><td valign="top" align="center" rowspan="1" colspan="1">60.12</td><td valign="top" align="center" rowspan="1" colspan="1">157,692</td><td valign="top" align="center" rowspan="1" colspan="1">88,420</td><td valign="top" align="center" rowspan="1" colspan="1">16,094</td><td valign="top" align="center" rowspan="1" colspan="1">26,589</td><td valign="top" align="center" rowspan="1" colspan="1">38.77</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>E. pseudowushanense</italic></td><td valign="top" align="center" rowspan="1" colspan="1">4,573,881</td><td valign="top" align="center" rowspan="1" colspan="1">461,961,981</td><td valign="top" align="center" rowspan="1" colspan="1">131,895</td><td valign="top" align="center" rowspan="1" colspan="1">2.88</td><td valign="top" align="center" rowspan="1" colspan="1">80.24</td><td valign="top" align="center" rowspan="1" colspan="1">157,168</td><td valign="top" align="center" rowspan="1" colspan="1">88,531</td><td valign="top" align="center" rowspan="1" colspan="1">17,069</td><td valign="top" align="center" rowspan="1" colspan="1">25,784</td><td valign="top" align="center" rowspan="1" colspan="1">38.77</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>E. koreanum</italic></td><td valign="top" align="center" rowspan="1" colspan="1">4,612,264</td><td valign="top" align="center" rowspan="1" colspan="1">465,838,664</td><td valign="top" align="center" rowspan="1" colspan="1">236,730</td><td valign="top" align="center" rowspan="1" colspan="1">5.10</td><td valign="top" align="center" rowspan="1" colspan="1">144.73</td><td valign="top" align="center" rowspan="1" colspan="1">157,218</td><td valign="top" align="center" rowspan="1" colspan="1">89,560</td><td valign="top" align="center" rowspan="1" colspan="1">17,222</td><td valign="top" align="center" rowspan="1" colspan="1">25,218</td><td valign="top" align="center" rowspan="1" colspan="1">38.72</td></tr></tbody></table></table-wrap></sec><sec><title>Genome features</title><p>The nucleotide sequences of the five <italic>Epimedium</italic> cp genomes ranged from 157,039 bp (<italic>E. acuminatum</italic>) to 159,112 bp (<italic>E. dolichostemon</italic>; Figure <xref ref-type="fig" rid="F1">1</xref>, Table <xref ref-type="table" rid="T1">1</xref>). All the five cp genomes displayed the typical quadripartite structure of angiosperms, which consisted of a pair of IR regions (25,218–27,741 bp) separated by a LSC region (86,561–89,560 bp), and a SSC region (16,094–17,222 bp). The average GC content was ~38.77%, which is almost identical with each other among the five complete <italic>Epimedium</italic> cp genomes.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p><bold>Gene maps of three <italic><bold>Epimedium</bold></italic>chloroplast genomes. (A)</bold><italic>E. acuminatum</italic>, <bold>(B)</bold>. <italic>E. dolichostemon</italic>, <bold>(C)</bold>. <italic>E. koreanum</italic>. Genes shown outside the outer circle are transcribed clockwise, and those inside are transcribed counterclockwise. Genes belonging to different functional groups are color coded. The dashed area in the inner circle indicates GC content of the chloroplast genomes.</p></caption><graphic xlink:href="fpls-07-00306-g0001"></graphic></fig><p>When duplicated genes in IR regions were counted only once, the five <italic>Epimedium</italic> cp genomes identically harbored 112 different genes arranged in the same order, including 78 protein-coding genes, 30 tRNA, and 4 rRNA. Twelve of the protein-coding genes and six of the tRNA genes contain introns, 15 of which contained a single intron, whereas, three have two introns (Table <xref ref-type="table" rid="T2">2</xref>). Among 78 protein-coding genes, 75 genes had the standard AUG as the initiator codon, but <italic>rps14</italic> and <italic>rps19</italic> started with GUG while <italic>rpl2</italic> and <italic>ndhD</italic> with ACG. An ACG codon may be restored to a canonical start codon (AUG) by RNA editing (Hoch et al., <xref rid="B8" ref-type="bibr">1991</xref>; Takenaka et al., <xref rid="B42" ref-type="bibr">2013</xref>), whereas, a GUG initiation codon has been reported in other cp genomes (Kuroda et al., <xref rid="B17" ref-type="bibr">2007</xref>; Gao et al., <xref rid="B6" ref-type="bibr">2009</xref>).</p><table-wrap id="T2" position="float"><label>Table 2</label><caption><p><bold>List of genes encoded by five <italic><bold>Epimedium</bold></italic> chloroplast genome</bold>.</p></caption><table frame="hsides" rules="groups"><thead><tr><th valign="top" align="left" rowspan="1" colspan="1"><bold>Category for genes</bold></th><th valign="top" align="left" rowspan="1" colspan="1"><bold>Group of genes</bold></th><th valign="top" align="left" rowspan="1" colspan="1"><bold>Name of genes</bold></th></tr></thead><tbody><tr><td valign="top" align="left" rowspan="1" colspan="1">Self-replication</td><td valign="top" align="left" rowspan="1" colspan="1">rRNA genes</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>rrn16</italic><xref ref-type="table-fn" rid="TN3"><sup>a</sup></xref>, <italic>rrn23</italic><xref ref-type="table-fn" rid="TN3"><sup>a</sup></xref>, <italic>rrn4.5</italic><xref ref-type="table-fn" rid="TN3"><sup>a</sup></xref>, <italic>rrn5</italic><xref ref-type="table-fn" rid="TN3"><sup>a</sup></xref></td></tr><tr><td rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">tRNA genes</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>trnA-UGC</italic><xref ref-type="table-fn" rid="TN1"><sup>*</sup></xref>,<xref ref-type="table-fn" rid="TN3"><sup>a</sup></xref>, <italic>trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnfM-CAU, trnG-GCC, trnG-UCC</italic><xref ref-type="table-fn" rid="TN1"><sup>*</sup></xref>, <italic>trnH-GUG, trnI-CAU</italic><xref ref-type="table-fn" rid="TN3"><sup>a</sup></xref>, <italic>trnI-GAU</italic>,<xref ref-type="table-fn" rid="TN3"><sup>a</sup></xref>, <italic>trnK-UUU</italic><xref ref-type="table-fn" rid="TN1"><sup>*</sup></xref>, <italic>trnL-CAA</italic><xref ref-type="table-fn" rid="TN3"><sup>a</sup></xref>, <italic>trnL-UAA</italic><xref ref-type="table-fn" rid="TN1"><sup>*</sup></xref>, <italic>trnL-UAG, trnM-CAU, trnN-GUU</italic><xref ref-type="table-fn" rid="TN3"><sup>a</sup></xref>, <italic>trnP-UGG, trnQ-UUG</italic><xref ref-type="table-fn" rid="TN3"><sup>a</sup></xref>, <italic>trnR-ACG</italic><xref ref-type="table-fn" rid="TN3"><sup>a</sup></xref>, <italic>trnR-UCU, trnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU, trnT-UGU, trnV-GAC</italic><xref ref-type="table-fn" rid="TN3"><sup>a</sup></xref>, <italic>trnV-UAC</italic><xref ref-type="table-fn" rid="TN1"><sup>*</sup></xref>, <italic>trnW-CCA, trnY-GUA</italic></td></tr><tr><td rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">Small subunit of ribosome</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>rps2, rps3, rps4, rps7</italic><xref ref-type="table-fn" rid="TN3"><sup>a</sup></xref>, <italic>rps8, rps11, rps12</italic><xref ref-type="table-fn" rid="TN2"><sup>**</sup></xref>,<xref ref-type="table-fn" rid="TN3"><sup>a</sup></xref>, <italic>rps14, rps15, rps16</italic><xref ref-type="table-fn" rid="TN1"><sup>*</sup></xref>, <italic>rps18, rps19</italic><xref ref-type="table-fn" rid="TN4"><sup>b</sup></xref></td></tr><tr><td rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">Large subunit of ribosome</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>rpl2</italic><xref ref-type="table-fn" rid="TN1"><sup>*</sup></xref>,<xref ref-type="table-fn" rid="TN4"><sup>b</sup></xref>, <italic>rpl14, rpl16</italic><xref ref-type="table-fn" rid="TN1"><sup>*</sup></xref>, <italic>rpl20, rpl22, rpl23</italic><xref ref-type="table-fn" rid="TN4"><sup>b</sup></xref>, <italic>rpl32, rpl33, rpl36</italic></td></tr><tr style="border-bottom: thin solid #000000;"><td rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">DNA dependent RNA polymerase</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>rpoA, rpoB, rpoC1</italic><xref ref-type="table-fn" rid="TN1"><sup>*</sup></xref>, <italic>rpoC2</italic></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Genes for phytosynthesis</td><td valign="top" align="left" rowspan="1" colspan="1">Subunits of NADH-dehydrogenase</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>ndhA</italic><xref ref-type="table-fn" rid="TN1"><sup>*</sup></xref>, <italic>ndhB</italic><xref ref-type="table-fn" rid="TN1"><sup>*</sup></xref>,<xref ref-type="table-fn" rid="TN3"><sup>a</sup></xref>, <italic>ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK</italic></td></tr><tr><td rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">Subunits of photosystem I</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>psaA, psaB, psaC, psaI, psaJ, ycf3</italic><xref ref-type="table-fn" rid="TN2"><sup>**</sup></xref></td></tr><tr><td rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">Subunits of photosystem II</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ</italic></td></tr><tr><td rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">Subunits of cytochrome b/f complex</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>petA, petB</italic><xref ref-type="table-fn" rid="TN1"><sup>*</sup></xref>, <italic>petD</italic><xref ref-type="table-fn" rid="TN1"><sup>*</sup></xref>, <italic>petG, petL, petN</italic></td></tr><tr><td rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">Subunits of ATP synthase</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>atpA, atpB, atpE, atpF</italic><xref ref-type="table-fn" rid="TN1"><sup>*</sup></xref>, <italic>atpH, atpI</italic></td></tr><tr style="border-bottom: thin solid #000000;"><td rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">Large subunit of rubisco</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>rbcL</italic></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Other genes</td><td valign="top" align="left" rowspan="1" colspan="1">Maturase</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>matK</italic></td></tr><tr><td rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">Protease</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>clpP</italic><xref ref-type="table-fn" rid="TN2"><sup>**</sup></xref></td></tr><tr><td rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">Envelope membrane protein</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>cemA</italic></td></tr><tr><td rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">Subunit of Acetyl-CoA-carboxylase</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>accD</italic></td></tr><tr style="border-bottom: thin solid #000000;"><td rowspan="1" colspan="1"></td><td valign="top" align="left" rowspan="1" colspan="1">c-type cytochrome synthesis gene</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>ccsA</italic></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Genes of unknown function</td><td valign="top" align="left" rowspan="1" colspan="1">Open Reading Frames (ORF, ycf)</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>ycf1, ycf2</italic><xref ref-type="table-fn" rid="TN3"><sup>a</sup></xref>, <italic>ycf4</italic></td></tr></tbody></table><table-wrap-foot><fn id="TN1"><label>*</label><p><italic>Gene with one intron</italic>,</p></fn><fn id="TN2"><label>**</label><p><italic>Gene with two introns</italic>,</p></fn><fn id="TN3"><label>a</label><p><italic>Gene with two copies</italic>,</p></fn><fn id="TN4"><label>b</label><p><italic>Gene with one or two copies</italic>.</p></fn></table-wrap-foot></table-wrap><p>The <italic>trnQ-UUG</italic> genes were duplicated in the LSC of the five <italic>Epimedium</italic> cp genomes and coherently separated by 101 bp with the same orientation. The nucleotide sequence of each copy was identical among the five <italic>Epimedium</italic> species. The length of one copy was 72 bp and the other with 73 bp, and the two copies were with 19% sequence divergence. The <italic>trnQ-UUG</italic> duplication had been reported in the family Geraniaceae (Weng et al., <xref rid="B45" ref-type="bibr">2013</xref>), but the gene duplication of <italic>Epimedium</italic> was firstly found in the basal eudicotyledons. Both MP and ML phylogenetic trees based on <italic>trnQ-UUG</italic> sequences of <italic>Epimedium</italic>, and other 11 basal eudicotyledons demonstrated that the two copies of the gene in <italic>Epimedium</italic> had most close relationship (Figure <xref ref-type="fig" rid="F2">2</xref>). This raised the possibility of independent duplications <italic>of trnQ-UUG</italic> in the genus <italic>Epimedium</italic>.</p><fig id="F2" position="float"><label>Figure 2</label><caption><p><bold>Phylogenetic trees constructed by <italic><bold>trnQ-UUG</bold></italic> sequences of <italic>Epimedium</italic>, and other 11 species of basal eudicotyledons with maximum parsimony (A) and maximum likelihood (B)</bold>. Numbers above node are bootstrap support values (>50%).</p></caption><graphic xlink:href="fpls-07-00306-g0002"></graphic></fig><p>The expansion and contraction of the IR region and the single-copy (SC) boundary regions was considered as a primarily mechanism causing the length variation of angiosperm cp genomes (Kim and Lee, <xref rid="B16" ref-type="bibr">2004</xref>). Although overall genomic structure including gene number and gene order were well-conserved, the five <italic>Epimedium</italic> cp genomes exhibited obvious different at the IR/SC boundary regions (Figure <xref ref-type="fig" rid="F3">3</xref>). The gene <italic>ycf1</italic> crossed the SSC/IRB region, and the pseudogene fragment ψ<italic>ycf1</italic> was located at the IRA region with 2181–3056 bp. The gene <italic>rpl22</italic> crossed the LSC/IRA region in <italic>E. acuminatum</italic>, and ψ<italic>rpl22</italic> with 296 bp was located at IRB region; <italic>rpl2</italic> crossed the LSC/IRA region in <italic>E. dolichostemon, E. lishihchenii</italic>, and <italic>E. pseudowushanense</italic>, and ψ<italic>rpl2</italic> with 226 bp was located at IRB region; <italic>rpl23</italic> crossed the LSC/IRA region in <italic>E. koreanum</italic>, and ψ<italic>rpl23</italic> with 33 bp was located at IRB region. At the junction of IRA/SSC region, the distance between ψ<italic>ycf1</italic> and <italic>ndhF</italic> ranged from 47 to 282 bp. At the junction of IRB/LSC region, the distance between ψ<italic>rpl22</italic> and <italic>trnH</italic> in <italic>E. acuminatum</italic> was 76 bp, the distance between ψ<italic>rpl2</italic> and <italic>trnH</italic> was from 71 to 76 bp in <italic>E. dolichostemon, E. lishihchenii</italic>, and <italic>E. pseudowushanense</italic>, and the distance between ψ<italic>rpl23 and trnH</italic> in <italic>E. koreanum</italic> was 92 bp. The variations at IR/SC boundary regions in the five <italic>Epimedium</italic> cp genomes led to their length variation of the four regions and whole genome sequences.</p><fig id="F3" position="float"><label>Figure 3</label><caption><p><bold>Comparisons of LSC, SSC, and IR region borders among the five <italic><bold>Epimedium</bold></italic> chloroplast genomes</bold>.</p></caption><graphic xlink:href="fpls-07-00306-g0003"></graphic></fig></sec><sec><title>Divergence hotspot regions</title><p>For purposes of the subsequent phylogenetic analyses and plant identification, the complete cp genomes of the five <italic>Epimedium</italic> species were compared and plotted using the mVISTA program to elucidate the level of sequence divergence (Figure <xref ref-type="fig" rid="F4">4</xref>). The IRs had lower sequence divergence than that in the SC regions, which also occurred in most higher plants and possibly due to copy correction between IR sequences by gene conversion (Khakhlova and Bock, <xref rid="B14" ref-type="bibr">2006</xref>). The whole genomes, protein-coding regions (pCDS), and non-coding regions (introns and spacers) exhibited divergence proportions of 3.97%, 1.10%, and 5.81%, respectively. For protein coding regions, <italic>rps16, psbK, rps132, rps14</italic>, and <italic>rps15</italic> had over 3% sequence divergences (Table <xref ref-type="supplementary-material" rid="SM4">S4</xref>). The non-coding regions had higher variability proportions, and four of the regions at the junction of the IRB and LSC had divergence proportions of 100% because of difference in expansion and contraction of IRB (Table <xref ref-type="supplementary-material" rid="SM5">S5</xref>). Fifty-two non-coding regions had variability proportions ranging from 3.03 to 86.55%, among which 17 regions, such as <italic>ycf1</italic>/<italic>ndhF, trnC-GCA</italic>/<italic>petN</italic>, and <italic>trnQ-UUG</italic>/<italic>psbK</italic>, had over 10% variability proportions. These divergence hotspot regions of the five <italic>Epimedium</italic> cp genome sequences provided abundant information for developing molecular markers for phylogenetic analyses and plant identification of <italic>Epimedium</italic> species.</p><fig id="F4" position="float"><label>Figure 4</label><caption><p><bold>Sequence identity plots among the five <italic><bold>Epimedium</bold></italic> chloroplast genomes</bold>.</p></caption><graphic xlink:href="fpls-07-00306-g0004"></graphic></fig></sec><sec><title>SSR polymorphisms</title><p>SSRs in the cp genome present high diversity in copy numbers, and are important molecular markers for plant population genetics, and evolutionary, and ecological studies (Huang et al., <xref rid="B9" ref-type="bibr">2014</xref>; Zhao et al., <xref rid="B53" ref-type="bibr">2015</xref>). With MISA analysis, 116 SSRs with a length of at least 10 bp were detected in the five <italic>Epimedium</italic> cp genomes with 103 loci showing polymorphism (Table <xref ref-type="table" rid="T3">3</xref>, Table <xref ref-type="supplementary-material" rid="SM6">S6</xref>, <xref ref-type="supplementary-material" rid="SM7">S7</xref>). Each <italic>Epimedium</italic> cp genome was found to contain 80 to 87 SSRs, of which 13 SSRs appeared same for the five cp genomes, and the numbers of polymorphic SSRs ranged from 67 to 74. Among the 116 SSRs, the mono-, di-, trin-, tetra-, penta-, and hexanucleotide SSRs were all detected, the mononucleotide SSRs were richest with a portion of 72.76%, and the mononucleotide A and T repeat units occupied the highest portion with 35.34% and 44.83%, respectively. These 116 SSR loci mainly located in intergenic spacer (IGS, 62.07%), following by pCDS (13.79%) and introns (23.28%). Only one SSR crossed the pCDS and IGS (<italic>psbI-psbI/trnS-GCU</italic>) in the cp genome of <italic>E. acuminatum</italic>. We observed that 16 SSRs located in 10 protein-coding genes [<italic>rpoc2, rpoB, psbC, psaA, psbF, ycf2</italic> (×4), <italic>ycf1</italic> (×4), <italic>rpl32, ndhE, ndhH</italic>] of the five <italic>Epimeidum</italic> cp genomes. Most of those SSR loci were located in LSC region, followed by SSC and IR regions. In general, the cp SSRs of the five <italic>Epimedium</italic> represented abundant variation, and undoubtedly useful for assays detecting polymorphisms at population-level as well as comparing more distantly phylogenetic relationships among <italic>Epimedium</italic> species.</p><table-wrap id="T3" position="float"><label>Table 3</label><caption><p><bold>Simple sequence repeats (SSRs) in the five <italic><bold>Epimedium</bold></italic> cp genomes</bold>.</p></caption><table frame="hsides" rules="groups"><thead><tr><th valign="top" align="left" rowspan="1" colspan="1"><bold>Species</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>SSR loci no.</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>PolyM. loci no.</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>PolyM. loci (%).</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>P1 loci no.</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>P2 loci no.</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>P3 loci no.</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>P4 loci no.</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>P5 loci no.</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>P6 loci no.</bold></th><th valign="top" align="center" colspan="4" style="border-bottom: thin solid #000000;" rowspan="1"><bold>Location</bold></th><th valign="top" align="center" colspan="3" style="border-bottom: thin solid #000000;" rowspan="1"><bold>Region</bold></th></tr><tr><th rowspan="1" colspan="1"></th><th rowspan="1" colspan="1"></th><th rowspan="1" colspan="1"></th><th rowspan="1" colspan="1"></th><th rowspan="1" colspan="1"></th><th rowspan="1" colspan="1"></th><th rowspan="1" colspan="1"></th><th rowspan="1" colspan="1"></th><th rowspan="1" colspan="1"></th><th rowspan="1" colspan="1"></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>IGS</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>Intron</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>pCDS</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>pCDS-IGS</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>LSC</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>SSC</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>IR</bold></th></tr></thead><tbody><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>E. acuminatum</italic></td><td valign="top" align="center" rowspan="1" colspan="1">87</td><td valign="top" align="center" rowspan="1" colspan="1">74</td><td valign="top" align="center" rowspan="1" colspan="1">85.06</td><td valign="top" align="center" rowspan="1" colspan="1">73</td><td valign="top" align="center" rowspan="1" colspan="1">6</td><td valign="top" align="center" rowspan="1" colspan="1">/</td><td valign="top" align="center" rowspan="1" colspan="1">5</td><td valign="top" align="center" rowspan="1" colspan="1">1</td><td valign="top" align="center" rowspan="1" colspan="1">2</td><td valign="top" align="center" rowspan="1" colspan="1">54</td><td valign="top" align="center" rowspan="1" colspan="1">19</td><td valign="top" align="center" rowspan="1" colspan="1">13</td><td valign="top" align="center" rowspan="1" colspan="1">1</td><td valign="top" align="center" rowspan="1" colspan="1">73</td><td valign="top" align="center" rowspan="1" colspan="1">10</td><td valign="top" align="center" rowspan="1" colspan="1">4</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>E. dolichostemon</italic></td><td valign="top" align="center" rowspan="1" colspan="1">80</td><td valign="top" align="center" rowspan="1" colspan="1">67</td><td valign="top" align="center" rowspan="1" colspan="1">83.75</td><td valign="top" align="center" rowspan="1" colspan="1">68</td><td valign="top" align="center" rowspan="1" colspan="1">6</td><td valign="top" align="center" rowspan="1" colspan="1">/</td><td valign="top" align="center" rowspan="1" colspan="1">5</td><td valign="top" align="center" rowspan="1" colspan="1">1</td><td valign="top" align="center" rowspan="1" colspan="1">/</td><td valign="top" align="center" rowspan="1" colspan="1">50</td><td valign="top" align="center" rowspan="1" colspan="1">18</td><td valign="top" align="center" rowspan="1" colspan="1">12</td><td valign="top" align="center" rowspan="1" colspan="1">/</td><td valign="top" align="center" rowspan="1" colspan="1">67</td><td valign="top" align="center" rowspan="1" colspan="1">9</td><td valign="top" align="center" rowspan="1" colspan="1">4</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>E. lishihchenii</italic></td><td valign="top" align="center" rowspan="1" colspan="1">87</td><td valign="top" align="center" rowspan="1" colspan="1">74</td><td valign="top" align="center" rowspan="1" colspan="1">85.06</td><td valign="top" align="center" rowspan="1" colspan="1">74</td><td valign="top" align="center" rowspan="1" colspan="1">6</td><td valign="top" align="center" rowspan="1" colspan="1">1</td><td valign="top" align="center" rowspan="1" colspan="1">6</td><td valign="top" align="center" rowspan="1" colspan="1">/</td><td valign="top" align="center" rowspan="1" colspan="1">/</td><td valign="top" align="center" rowspan="1" colspan="1">51</td><td valign="top" align="center" rowspan="1" colspan="1">22</td><td valign="top" align="center" rowspan="1" colspan="1">14</td><td valign="top" align="center" rowspan="1" colspan="1">/</td><td valign="top" align="center" rowspan="1" colspan="1">71</td><td valign="top" align="center" rowspan="1" colspan="1">12</td><td valign="top" align="center" rowspan="1" colspan="1">4</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>E. pseudowushanense</italic></td><td valign="top" align="center" rowspan="1" colspan="1">84</td><td valign="top" align="center" rowspan="1" colspan="1">71</td><td valign="top" align="center" rowspan="1" colspan="1">84.52</td><td valign="top" align="center" rowspan="1" colspan="1">71</td><td valign="top" align="center" rowspan="1" colspan="1">6</td><td valign="top" align="center" rowspan="1" colspan="1">/</td><td valign="top" align="center" rowspan="1" colspan="1">6</td><td valign="top" align="center" rowspan="1" colspan="1">1</td><td valign="top" align="center" rowspan="1" colspan="1">/</td><td valign="top" align="center" rowspan="1" colspan="1">50</td><td valign="top" align="center" rowspan="1" colspan="1">21</td><td valign="top" align="center" rowspan="1" colspan="1">13</td><td valign="top" align="center" rowspan="1" colspan="1">/</td><td valign="top" align="center" rowspan="1" colspan="1">71</td><td valign="top" align="center" rowspan="1" colspan="1">9</td><td valign="top" align="center" rowspan="1" colspan="1">4</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>E. koreanum</italic></td><td valign="top" align="center" rowspan="1" colspan="1">85</td><td valign="top" align="center" rowspan="1" colspan="1">72</td><td valign="top" align="center" rowspan="1" colspan="1">84.71</td><td valign="top" align="center" rowspan="1" colspan="1">72</td><td valign="top" align="center" rowspan="1" colspan="1">7</td><td valign="top" align="center" rowspan="1" colspan="1">/</td><td valign="top" align="center" rowspan="1" colspan="1">5</td><td valign="top" align="center" rowspan="1" colspan="1">/</td><td valign="top" align="center" rowspan="1" colspan="1">1</td><td valign="top" align="center" rowspan="1" colspan="1">49</td><td valign="top" align="center" rowspan="1" colspan="1">23</td><td valign="top" align="center" rowspan="1" colspan="1">13</td><td valign="top" align="center" rowspan="1" colspan="1">/</td><td valign="top" align="center" rowspan="1" colspan="1">70</td><td valign="top" align="center" rowspan="1" colspan="1">11</td><td valign="top" align="center" rowspan="1" colspan="1">4</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Total Loci</td><td valign="top" align="center" rowspan="1" colspan="1">116</td><td valign="top" align="center" rowspan="1" colspan="1">103</td><td valign="top" align="center" rowspan="1" colspan="1">88.79</td><td valign="top" align="center" rowspan="1" colspan="1">96</td><td valign="top" align="center" rowspan="1" colspan="1">7</td><td valign="top" align="center" rowspan="1" colspan="1">1</td><td valign="top" align="center" rowspan="1" colspan="1">8</td><td valign="top" align="center" rowspan="1" colspan="1">1</td><td valign="top" align="center" rowspan="1" colspan="1">3</td><td valign="top" align="center" rowspan="1" colspan="1">72</td><td valign="top" align="center" rowspan="1" colspan="1">27</td><td valign="top" align="center" rowspan="1" colspan="1">16</td><td valign="top" align="center" rowspan="1" colspan="1">1</td><td valign="top" align="center" rowspan="1" colspan="1">97</td><td valign="top" align="center" rowspan="1" colspan="1">13</td><td valign="top" align="center" rowspan="1" colspan="1">6</td></tr></tbody></table><table-wrap-foot><p><italic>P1 to P6 represented SSR loci with mono-, di-, tri-, tetra-, penta-, and hexanucleotide repeats, respectively</italic>.</p></table-wrap-foot></table-wrap></sec><sec><title>Repetitive sequences</title><p>With the criterion of copy size 30 bp or longer and sequence identity >90%, REPuter identified a total of 49 repeats in the five <italic>Epimedium</italic> cp genomes, including direct, and palindromic repeats (Figure <xref ref-type="fig" rid="F5">5</xref>, Table <xref ref-type="supplementary-material" rid="SM8">S8</xref>). Except for <italic>E. koreanum</italic> with 24 direct repeats and 25 palindromic repeats, the other four <italic>Epimedium</italic> species identically possessed 23 direct repeats, and 26 palindromic repeats. The lengths of repeats in the five <italic>Epimedium</italic> cp genomes ranged from 31 to 131 bp, and the copy lengths with 30–49 bp are most common (61.22%) while those with more than 100 bp were least (7.76%). Under the criterion with identical lengths located in homologous regions as shared repeats, we investigated the repeats shared among the five <italic>Epimedium</italic> cp genomes. There were 16 repeats shared by the five <italic>Epimedium</italic> cp genomes, 15 repeats shared by the four <italic>Epimedium</italic> species endemic to China, 13 repeats shared by <italic>E. koreanum</italic> and the three of four Chinese <italic>Epimedium</italic> species, and four repeats shared by two or three Chinese <italic>Epimedium</italic> species. <italic>E. koreanum</italic> had the most unique repeats (20), followed with <italic>E. acuminatum</italic> (16), while the other three <italic>Epimedium</italic> species had one to three unique repeats. The repeats of the five <italic>Epimedium</italic> cp genomes were mainly located in pCDS and IGS, while the minority was located in intron and <italic>rrn</italic> gene coding region (rCDS), or covered across IGS and one of pCDS, rCDS, or <italic>trn</italic> gene coding region (tCDS). Except for <italic>E. koreanum</italic>, the proportions of repeat locations were identical in the other four <italic>Epimedium</italic> species.</p><fig id="F5" position="float"><label>Figure 5</label><caption><p><bold>Analysis of repeated sequences in the five <italic><bold>Epimedium</bold></italic> chloroplast genomes. (A)</bold> Frequency of the direct repeats by length; <bold>(B)</bold>. Frequency of the palindromic repeats; <bold>(C)</bold>. Location of repeats in the four <italic>Epimedium</italic> cp genomes endemic to China; <bold>(D)</bold>. Location of repeats in the cp genome of <italic>E. koreanum</italic>; <bold>(E)</bold>. Summary of shared repeats among the five <italic>Epimedium</italic> chloroplast genomes. chlo, <italic>E. acuminatum</italic>; dewu, <italic>E. dolichostemon</italic>; lish, <italic>E. lishihchenii</italic>; pseu, <italic>E. pseudowushanense</italic>; kore, <italic>E. koreanum</italic>; IGS, intergenic spacer.</p></caption><graphic xlink:href="fpls-07-00306-g0005"></graphic></fig><p>Contrasting to the major repeats of most angiosperm plant cp genomes located in noncoding regions (Uthaipaisanwong et al., <xref rid="B44" ref-type="bibr">2012</xref>; Yao et al., <xref rid="B48" ref-type="bibr">2015</xref>), the proportions of repeats located in coding regions (CDS) were higher than those in noncoding regions in <italic>Epimedium</italic> species. In <italic>E. koreanum</italic> cp genomes, the proportion of the repeats located in pCDS led to 63.27%, while the repeats located in IGS only accounted for 22.45%. Previous work suggested that repeat sequences have played an important role in sequence rearranging and variation in cp genomes through illegitimate recombination and slipped-strand mispairing (Bausher et al., <xref rid="B2" ref-type="bibr">2006</xref>; Saski et al., <xref rid="B36" ref-type="bibr">2007</xref>; Huang et al., <xref rid="B9" ref-type="bibr">2014</xref>). Our research also showed that divergent regions of cp genomes were associated with various repeat sequences such as <italic>ycf1</italic> gene and intergenic <italic>trnQ-UUG</italic>/<italic>psbK</italic>. These repeats may further serve as genetic markers for phylogenetic and population genetic studies on <italic>Epimedium</italic> species.</p></sec><sec><title>Phylogenetic analysis</title><p>The cp genome sequences are addressed successfully for the phylogenetic studies of angiosperm (Jansen et al., <xref rid="B10" ref-type="bibr">2007</xref>; Huang et al., <xref rid="B9" ref-type="bibr">2014</xref>; Kim et al., <xref rid="B15" ref-type="bibr">2015</xref>). In the present studies, three datasets (protein coding exons, introns and spacers, and whole complete cp genome sequences) from cp genomes of five <italic>Epimedium</italic> species and two outgroups were used to perform phylogenetic analysis. Among the three datasets, introns and spacers contained the highest parsimony informative characters (6.85%), followed by whole complete cp genome sequences (5.22%) and protein coding exons (5.03%). Using MP and ML analyses, phylogenetic trees were built based on the three datasets (Figure <xref ref-type="fig" rid="F6">6</xref>). The topologies based on both analyses were highly concordant in each dataset, as well as the dendrograms based on the noncoding sequences and whole complete cp genome sequences, and the phylogenetic trees of the three datasets were largely congruent with each other. For the five <italic>Epimedium</italic> species, <italic>E. koreanum</italic> is distributed in Northeast China, Japan, and Korea, and belongs to sect. <italic>Macroceras</italic>, while the other four species are native to Central and Southwest China, being attributed to sect. <italic>Diphyllon</italic> (Stearn, <xref rid="B39" ref-type="bibr">2002</xref>). The resulting six phylogenetic trees identically exhibited that <italic>E. koreanum</italic> were firstly separated from the other four <italic>Epimedium</italic> species. For the four <italic>Epimedium</italic> species of sect. <italic>Diphyllon, E. dolichostemon</italic> has relatively small flowers and short spurs, being a member of ser. <italic>Brachycerae</italic>; the other three species has large flowers with petals bearing long spurs, of which <italic>E. acuminatum</italic> and <italic>E. lishihchenii</italic> possess petal without basal laminae, being attributed to ser. <italic>Dolichocerae</italic>, while <italic>E. pseudowushanense</italic> possesses petal with slight basal lamina, belonging to ser. <italic>Davidianae</italic>. In accordance with classical taxonomy of <italic>Epimedium</italic> (Stearn, <xref rid="B39" ref-type="bibr">2002</xref>), phylogenetic trees based on noncoding regions and whole complete cp genome sequences all supported that <italic>E. dolichostemon</italic> was early divided from the other three species of sect. <italic>Diphyllon</italic>. However, the basal position of <italic>E. dolichostemon</italic> among four species of sec. <italic>Diphyllon</italic> was inconsistent with Stearn's (<xref rid="B39" ref-type="bibr">2002</xref>) and Ying's (<xref rid="B49" ref-type="bibr">2002</xref>) interpretation on floral evolution of the genus. Furthermore, all trees based on the three datasets identically supported that <italic>E. lishihchenii</italic> firstly clustered with <italic>E. pseudowushanense</italic>, not with <italic>E. acuminatum</italic> from the same series, which were coincident with the previous phylogenetic studies based on AFLPs (Zhang et al., <xref rid="B52" ref-type="bibr">2014</xref>). These results showed that Stearn's (<xref rid="B39" ref-type="bibr">2002</xref>) taxonomic system of <italic>Epimedium</italic> is reasonable on the whole and the phylogenetic relationships within Chinese sect. <italic>Diphyllon</italic> are closely related with corolla characters, especially with petals. However, the evolutionary relationships and the divisions within the section need further investigation applying more evidences.</p><fig id="F6" position="float"><label>Figure 6</label><caption><p><bold>Phylogenetic relationships of the five <italic><bold>Epimedium</bold></italic> species constructed by CDS regions (A), noncoding regions (B), and whole cp genome sequences (C) with maximum parsimony (MP) and maximum likelihood</bold>. Numbers above node are bootstrap support values (>50%) with MP bootstrap values on the left and ML bootstrap on the right.</p></caption><graphic xlink:href="fpls-07-00306-g0006"></graphic></fig></sec></sec><sec id="s4"><title>Author contributions</title><p>YZ, YW, and TY conceived and designed the experiment, and writed the paper. JC, WZ, AL, KK, and SL collected the materials. KK, SL, YZ, and LD performed the experiments. KK and SL completed the sequence assembly. LD, YZ, LW, and WH conducted the comprehensive analyses on the cp genome sequences.</p><sec><title>Conflict of interest statement</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></sec></body><back><ack><p>This research is supported by the National Natural Science Foundation of China (30900076) and Key Research Program of the Chinese Academy of Sciences (KSZD-EW-Z-004), and by the Bio & Medical Technology Development Program of the NRF funded by the Korean government, MSIP (NRF-2015M3A9A5030733). We thank Jia Li and Ke Tao for their help on data analysis, and Lei Gao and Bo Wang for their valuable comments on the manuscript.</p></ack><sec sec-type="supplementary-material" id="s5"><title>Supplementary material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type="uri" xlink:href="http://journal.frontiersin.org/article/10.3389/fpls.2016.00306">http://journal.frontiersin.org/article/10.3389/fpls.2016.00306</ext-link></p><supplementary-material content-type="local-data" id="SM1"><media xlink:href="Table1.DOCX"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="SM2"><media xlink:href="Table2.DOCX"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="SM3"><media xlink:href="Table3.DOCX"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="SM4"><media xlink:href="Table4.docx"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="SM5"><media xlink:href="Table5.docx"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="SM6"><media xlink:href="Table6.DOCX"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="SM7"><media xlink:href="Table7.DOCX"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="SM8"><media xlink:href="Table8.DOCX"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id="B1"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Avent</surname><given-names>T.</given-names></name></person-group> (<year>2010</year>). <article-title>An overview of <italic>Epimedium</italic></article-title>. <source>Plantsman</source><volume>9</volume>, <fpage>10</fpage>–<lpage>17</lpage>. 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