Evolution of the bHLH Genes Involved in Stomatal Development: Implications for the Expansion of Developmental Complexity of Stomata in Land Plants
Identifieur interne : 001175 ( Pmc/Corpus ); précédent : 001174; suivant : 001176Evolution of the bHLH Genes Involved in Stomatal Development: Implications for the Expansion of Developmental Complexity of Stomata in Land Plants
Auteurs : Jin-Hua Ran ; Ting-Ting Shen ; Wen-Juan Liu ; Xiao-Quan WangSource :
- PLoS ONE [ 1932-6203 ] ; 2013.
Abstract
Stomata play significant roles in plant evolution. A trio of closely related basic Helix-Loop-Helix (bHLH) subgroup Ia genes,
Url:
DOI: 10.1371/journal.pone.0078997
PubMed: 24244399
PubMed Central: 3823973
Links to Exploration step
PMC:3823973Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Evolution of the bHLH Genes Involved in Stomatal Development: Implications for the Expansion of Developmental Complexity of Stomata in Land Plants</title><author><name sortKey="Ran, Jin Hua" sort="Ran, Jin Hua" uniqKey="Ran J" first="Jin-Hua" last="Ran">Jin-Hua Ran</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Shen, Ting Ting" sort="Shen, Ting Ting" uniqKey="Shen T" first="Ting-Ting" last="Shen">Ting-Ting Shen</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Liu, Wen Juan" sort="Liu, Wen Juan" uniqKey="Liu W" first="Wen-Juan" last="Liu">Wen-Juan Liu</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Wang, Xiao Quan" sort="Wang, Xiao Quan" uniqKey="Wang X" first="Xiao-Quan" last="Wang">Xiao-Quan Wang</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">24244399</idno><idno type="pmc">3823973</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3823973</idno><idno type="RBID">PMC:3823973</idno><idno type="doi">10.1371/journal.pone.0078997</idno><date when="2013">2013</date><idno type="wicri:Area/Pmc/Corpus">001175</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Evolution of the bHLH Genes Involved in Stomatal Development: Implications for the Expansion of Developmental Complexity of Stomata in Land Plants</title><author><name sortKey="Ran, Jin Hua" sort="Ran, Jin Hua" uniqKey="Ran J" first="Jin-Hua" last="Ran">Jin-Hua Ran</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Shen, Ting Ting" sort="Shen, Ting Ting" uniqKey="Shen T" first="Ting-Ting" last="Shen">Ting-Ting Shen</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Liu, Wen Juan" sort="Liu, Wen Juan" uniqKey="Liu W" first="Wen-Juan" last="Liu">Wen-Juan Liu</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Wang, Xiao Quan" sort="Wang, Xiao Quan" uniqKey="Wang X" first="Xiao-Quan" last="Wang">Xiao-Quan Wang</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author></analytic><series><title level="j">PLoS ONE</title><idno type="eISSN">1932-6203</idno><imprint><date when="2013">2013</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Stomata play significant roles in plant evolution. A trio of closely related basic Helix-Loop-Helix (bHLH) subgroup Ia genes, <italic>SPCH</italic>, <italic>MUTE</italic> and <italic>FAMA</italic>, mediate sequential steps of stomatal development, and their functions may be conserved in land plants. However, the evolutionary history of the putative <italic>SPCH/MUTE/FAMA</italic> genes is still greatly controversial, especially the phylogenetic positions of the bHLH Ia members from basal land plants. To better understand the evolutionary pattern and functional diversity of the bHLH genes involved in stomatal development, we made a comprehensive evolutionary analysis of the homologous genes from 54 species representing the major lineages of green plants. The phylogenetic analysis indicated: (1) All bHLH Ia genes from the two basal land plants <italic>Physcomitrella</italic> and <italic>Selaginella</italic> were closely related to the <italic>FAMA</italic> genes of seed plants; and (2) the gymnosperm ‘<italic>SPCH</italic>’ genes were sister to a clade comprising the angiosperm <italic>SPCH</italic> and <italic>MUTE</italic> genes, while the <italic>FAMA</italic> genes of gymnosperms and angiosperms had a sister relationship. The revealed phylogenetic relationships are also supported by the distribution of gene structures and previous functional studies. Therefore, we deduce that the function of <italic>FAMA</italic> might be ancestral in the bHLH Ia subgroup. In addition, the gymnosperm “<italic>SPCH”</italic> genes may represent an ancestral state and have a dual function of <italic>SPCH</italic> and <italic>MUTE</italic>, two genes that could have originated from a duplication event in the common ancestor of angiosperms. Moreover, in angiosperms, <italic>SPCHs</italic> have experienced more duplications and harbor more copies than <italic>MUTEs</italic> and <italic>FAMAs</italic>, which, together with variation of the stomatal development in the entry division, implies that <italic>SPCH</italic> might have contributed greatly to the diversity of stomatal development. Based on the above, we proposed a model for the correlation between the evolution of stomatal development and the genes involved in this developmental process in land plants.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Bateman, Rm" uniqKey="Bateman R">RM Bateman</name></author><author><name sortKey="Crane, Pr" uniqKey="Crane P">PR Crane</name></author><author><name sortKey="Dimichele, Wa" uniqKey="Dimichele W">WA DiMichele</name></author><author><name sortKey="Kenrick, Pr" uniqKey="Kenrick P">PR Kenrick</name></author><author><name sortKey="Rowe, Np" uniqKey="Rowe N">NP Rowe</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Delaux, Pm" uniqKey="Delaux P">PM Delaux</name></author><author><name sortKey="Nanda, Ak" uniqKey="Nanda A">AK Nanda</name></author><author><name sortKey="Mathe, C" uniqKey="Mathe C">C Mathe</name></author><author><name sortKey="Sejalon Delmas, N" uniqKey="Sejalon Delmas N">N Sejalon-Delmas</name></author><author><name sortKey="Dunand, C" uniqKey="Dunand C">C Dunand</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Edwards, D" uniqKey="Edwards D">D Edwards</name></author><author><name sortKey="Kerp, H" uniqKey="Kerp H">H Kerp</name></author><author><name sortKey="Hass, H" uniqKey="Hass H">H Hass</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Raven, Ja" uniqKey="Raven J">JA Raven</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sack, Fd" uniqKey="Sack F">FD Sack</name></author><author><name sortKey="Paolillo, Dj" uniqKey="Paolillo D">DJ Paolillo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Torii, Ku" uniqKey="Torii K">KU Torii</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Beerling, Dj" uniqKey="Beerling D">DJ Beerling</name></author><author><name sortKey="Franks, Pj" uniqKey="Franks P">PJ Franks</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chater, C" uniqKey="Chater C">C Chater</name></author><author><name sortKey="Kamisugi, Y" uniqKey="Kamisugi Y">Y Kamisugi</name></author><author><name sortKey="Movahedi, M" uniqKey="Movahedi M">M Movahedi</name></author><author><name sortKey="Fleming, A" uniqKey="Fleming A">A Fleming</name></author><author><name sortKey="Cuming, Ac" uniqKey="Cuming A">AC Cuming</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Haworth, M" uniqKey="Haworth M">M Haworth</name></author><author><name sortKey="Elliott Kingston, C" uniqKey="Elliott Kingston C">C Elliott-Kingston</name></author><author><name sortKey="Mcelwain, Jc" uniqKey="Mcelwain J">JC McElwain</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mcadam, Sam" uniqKey="Mcadam S">SAM McAdam</name></author><author><name sortKey="Brodribb, Tj" uniqKey="Brodribb T">TJ Brodribb</name></author><author><name sortKey="Ross, Jj" uniqKey="Ross J">JJ Ross</name></author><author><name sortKey="Jordan, Gj" uniqKey="Jordan G">GJ Jordan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Doi, M" uniqKey="Doi M">M Doi</name></author><author><name sortKey="Shimazaki, K" uniqKey="Shimazaki K">K Shimazaki</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Brodribb, Tj" uniqKey="Brodribb T">TJ Brodribb</name></author><author><name sortKey="Mcadam, Sam" uniqKey="Mcadam S">SAM McAdam</name></author><author><name sortKey="Jordan, Gj" uniqKey="Jordan G">GJ Jordan</name></author><author><name sortKey="Feild, Ts" uniqKey="Feild T">TS Feild</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Brodribb, Tj" uniqKey="Brodribb T">TJ Brodribb</name></author><author><name sortKey="Mcadam, Sam" uniqKey="Mcadam S">SAM McAdam</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mcadam, Sa" uniqKey="Mcadam S">SA McAdam</name></author><author><name sortKey="Brodribb, Tj" uniqKey="Brodribb T">TJ Brodribb</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="De Boer, Hj" uniqKey="De Boer H">HJ de Boer</name></author><author><name sortKey="Eppinga, Mb" uniqKey="Eppinga M">MB Eppinga</name></author><author><name sortKey="Wassen, Mj" uniqKey="Wassen M">MJ Wassen</name></author><author><name sortKey="Dekker, Sc" uniqKey="Dekker S">SC Dekker</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Robinson, Jm" uniqKey="Robinson J">JM Robinson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Franks, Pj" uniqKey="Franks P">PJ Franks</name></author><author><name sortKey="Beerling, Dj" uniqKey="Beerling D">DJ Beerling</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Doi, M" uniqKey="Doi M">M Doi</name></author><author><name sortKey="Wada, M" uniqKey="Wada M">M Wada</name></author><author><name sortKey="Shimazaki, K" uniqKey="Shimazaki K">K Shimazaki</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Croxdale, Jl" uniqKey="Croxdale J">JL Croxdale</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Darwin, F" uniqKey="Darwin F">F Darwin</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Berry, Ja" uniqKey="Berry J">JA Berry</name></author><author><name sortKey="Beerling, Dj" uniqKey="Beerling D">DJ Beerling</name></author><author><name sortKey="Franks, Pj" uniqKey="Franks P">PJ Franks</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ruszala, Em" uniqKey="Ruszala E">EM Ruszala</name></author><author><name sortKey="Beerling, Dj" uniqKey="Beerling D">DJ Beerling</name></author><author><name sortKey="Franks, Pj" uniqKey="Franks P">PJ Franks</name></author><author><name sortKey="Chater, C" uniqKey="Chater C">C Chater</name></author><author><name sortKey="Casson, Sa" uniqKey="Casson S">SA Casson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mcadam, Sa" uniqKey="Mcadam S">SA McAdam</name></author><author><name sortKey="Brodribb, Tj" uniqKey="Brodribb T">TJ Brodribb</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kanaoka, Mm" uniqKey="Kanaoka M">MM Kanaoka</name></author><author><name sortKey="Pillitteri, Lj" uniqKey="Pillitteri L">LJ Pillitteri</name></author><author><name sortKey="Fujii, H" uniqKey="Fujii H">H Fujii</name></author><author><name sortKey="Yoshida, Y" uniqKey="Yoshida Y">Y Yoshida</name></author><author><name sortKey="Bogenschutz, Nl" uniqKey="Bogenschutz N">NL Bogenschutz</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Pillitteri, Lj" uniqKey="Pillitteri L">LJ Pillitteri</name></author><author><name sortKey="Torii, Ku" uniqKey="Torii K">KU Torii</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Macalister, Ca" uniqKey="Macalister C">CA MacAlister</name></author><author><name sortKey="Ohashi Ito, K" uniqKey="Ohashi Ito K">K Ohashi-Ito</name></author><author><name sortKey="Bergmann, Dc" uniqKey="Bergmann D">DC Bergmann</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ohashi Ito, K" uniqKey="Ohashi Ito K">K Ohashi-Ito</name></author><author><name sortKey="Bergmann, Dc" uniqKey="Bergmann D">DC Bergmann</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gudesblat, Ge" uniqKey="Gudesblat G">GE Gudesblat</name></author><author><name sortKey="Schneider Pizon, J" uniqKey="Schneider Pizon J">J Schneider-Pizon</name></author><author><name sortKey="Betti, C" uniqKey="Betti C">C Betti</name></author><author><name sortKey="Mayerhofer, J" uniqKey="Mayerhofer J">J Mayerhofer</name></author><author><name sortKey="Vanhoutte, I" uniqKey="Vanhoutte I">I Vanhoutte</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Pires, N" uniqKey="Pires N">N Pires</name></author><author><name sortKey="Dolan, L" uniqKey="Dolan L">L Dolan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Heim, Ma" uniqKey="Heim M">MA Heim</name></author><author><name sortKey="Jakoby, M" uniqKey="Jakoby M">M Jakoby</name></author><author><name sortKey="Werber, M" uniqKey="Werber M">M Werber</name></author><author><name sortKey="Martin, C" uniqKey="Martin C">C Martin</name></author><author><name sortKey="Weisshaar, B" uniqKey="Weisshaar B">B Weisshaar</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Macalister, Ca" uniqKey="Macalister C">CA MacAlister</name></author><author><name sortKey="Bergmann, Dc" uniqKey="Bergmann D">DC Bergmann</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Vaten, A" uniqKey="Vaten A">A Vaten</name></author><author><name sortKey="Bergmann, Dc" uniqKey="Bergmann D">DC Bergmann</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Peterson, Km" uniqKey="Peterson K">KM Peterson</name></author><author><name sortKey="Rychel, Al" uniqKey="Rychel A">AL Rychel</name></author><author><name sortKey="Torii, Ku" uniqKey="Torii K">KU Torii</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Toledo Ortiz, G" uniqKey="Toledo Ortiz G">G Toledo-Ortiz</name></author><author><name sortKey="Huq, E" uniqKey="Huq E">E Huq</name></author><author><name sortKey="Quail, Ph" uniqKey="Quail P">PH Quail</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Li, X" uniqKey="Li X">X Li</name></author><author><name sortKey="Duan, X" uniqKey="Duan X">X Duan</name></author><author><name sortKey="Jiang, H" uniqKey="Jiang H">H Jiang</name></author><author><name sortKey="Sun, Y" uniqKey="Sun Y">Y Sun</name></author><author><name sortKey="Tang, Y" uniqKey="Tang Y">Y Tang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Doyle, Ja" uniqKey="Doyle J">JA Doyle</name></author><author><name sortKey="Doyle, Jl" uniqKey="Doyle J">JL Doyle</name></author></analytic></biblStruct><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Guo, D" uniqKey="Guo D">D–M Guo</name></author><author><name sortKey="Ran, J" uniqKey="Ran J">J–H Ran</name></author><author><name sortKey="Wang, X Q" uniqKey="Wang X">X-Q Wang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ran, J" uniqKey="Ran J">J–H Ran</name></author><author><name sortKey="Gao, H" uniqKey="Gao H">H Gao</name></author><author><name sortKey="Wang, X Q" uniqKey="Wang X">X-Q Wang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Thompson, Jd" uniqKey="Thompson J">JD Thompson</name></author><author><name sortKey="Gibson, Tj" uniqKey="Gibson T">TJ Gibson</name></author><author><name sortKey="Plewniak, F" uniqKey="Plewniak F">F Plewniak</name></author><author><name sortKey="Jeanmougin, F" uniqKey="Jeanmougin F">F Jeanmougin</name></author><author><name sortKey="Higgins, Dg" uniqKey="Higgins D">DG Higgins</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Xia, X" uniqKey="Xia X">X Xia</name></author><author><name sortKey="Xie, Z" uniqKey="Xie Z">Z Xie</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Darriba, D" uniqKey="Darriba D">D Darriba</name></author><author><name sortKey="Taboada, Gl" uniqKey="Taboada G">GL Taboada</name></author><author><name sortKey="Doallo, R" uniqKey="Doallo R">R Doallo</name></author><author><name sortKey="Posada, D" uniqKey="Posada D">D Posada</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Darriba, D" uniqKey="Darriba D">D Darriba</name></author><author><name sortKey="Taboada, Gl" uniqKey="Taboada G">GL Taboada</name></author><author><name sortKey="Doallo, R" uniqKey="Doallo R">R Doallo</name></author><author><name sortKey="Posada, D" uniqKey="Posada D">D Posada</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Huelsenbeck, Jp" uniqKey="Huelsenbeck J">JP Huelsenbeck</name></author><author><name sortKey="Ronquist, F" uniqKey="Ronquist F">F Ronquist</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Guindon, S" uniqKey="Guindon S">S Guindon</name></author><author><name sortKey="Gascuel, O" uniqKey="Gascuel O">O Gascuel</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Guindon, S" uniqKey="Guindon S">S Guindon</name></author><author><name sortKey="Rodrigo, Ag" uniqKey="Rodrigo A">AG Rodrigo</name></author><author><name sortKey="Dyer, Ka" uniqKey="Dyer K">KA Dyer</name></author><author><name sortKey="Huelsenbeck, Jp" uniqKey="Huelsenbeck J">JP Huelsenbeck</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Yang, Z" uniqKey="Yang Z">Z Yang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Librado, P" uniqKey="Librado P">P Librado</name></author><author><name sortKey="Rozas, J" uniqKey="Rozas J">J Rozas</name></author></analytic></biblStruct><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Liu, T" uniqKey="Liu T">T Liu</name></author><author><name sortKey="Ohashi Ito, K" uniqKey="Ohashi Ito K">K Ohashi-Ito</name></author><author><name sortKey="Bergmann, Dc" uniqKey="Bergmann D">DC Bergmann</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lampard, Gr" uniqKey="Lampard G">GR Lampard</name></author><author><name sortKey="Macalister, Ca" uniqKey="Macalister C">CA MacAlister</name></author><author><name sortKey="Bergmann, Dc" uniqKey="Bergmann D">DC Bergmann</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Nystedt, B" uniqKey="Nystedt B">B Nystedt</name></author><author><name sortKey="Street, Nr" uniqKey="Street N">NR Street</name></author><author><name sortKey="Wetterbom, A" uniqKey="Wetterbom A">A Wetterbom</name></author><author><name sortKey="Zuccolo, A" uniqKey="Zuccolo A">A Zuccolo</name></author><author><name sortKey="Lin, Yc" uniqKey="Lin Y">YC Lin</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Pillitteri, Lj" uniqKey="Pillitteri L">LJ Pillitteri</name></author><author><name sortKey="Torii, Ku" uniqKey="Torii K">KU Torii</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Payne, Ww" uniqKey="Payne W">WW Payne</name></author></analytic></biblStruct><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Apostolakos, P" uniqKey="Apostolakos P">P Apostolakos</name></author><author><name sortKey="Panteris, E" uniqKey="Panteris E">E Panteris</name></author><author><name sortKey="Galatis, B" uniqKey="Galatis B">B Galatis</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Johnson, Rw" uniqKey="Johnson R">RW Johnson</name></author><author><name sortKey="Riding, Rt" uniqKey="Riding R">RT Riding</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rudall, Pj" uniqKey="Rudall P">PJ Rudall</name></author><author><name sortKey="Rowland, A" uniqKey="Rowland A">A Rowland</name></author><author><name sortKey="Bateman, Rm" uniqKey="Bateman R">RM Bateman</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Carpenter, Kj" uniqKey="Carpenter K">KJ Carpenter</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sack, Fd" uniqKey="Sack F">FD Sack</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Stebbins, Gl" uniqKey="Stebbins G">GL Stebbins</name></author><author><name sortKey="Jain, Sk" uniqKey="Jain S">SK Jain</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">PLoS One</journal-id><journal-id journal-id-type="iso-abbrev">PLoS ONE</journal-id><journal-id journal-id-type="publisher-id">plos</journal-id><journal-id journal-id-type="pmc">plosone</journal-id><journal-title-group><journal-title>PLoS ONE</journal-title></journal-title-group><issn pub-type="epub">1932-6203</issn><publisher><publisher-name>Public Library of Science</publisher-name><publisher-loc>San Francisco, USA</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">24244399</article-id><article-id pub-id-type="pmc">3823973</article-id><article-id pub-id-type="publisher-id">PONE-D-13-32757</article-id><article-id pub-id-type="doi">10.1371/journal.pone.0078997</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group></article-categories><title-group><article-title>Evolution of the bHLH Genes Involved in Stomatal Development: Implications for the Expansion of Developmental Complexity of Stomata in Land Plants</article-title><alt-title alt-title-type="running-head">Evolution of the bHLH Genes in Stomata Development</alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Ran</surname><given-names>Jin-Hua</given-names></name><xref ref-type="aff" rid="aff1"></xref><xref ref-type="corresp" rid="cor1"><sup>*</sup></xref></contrib><contrib contrib-type="author"><name><surname>Shen</surname><given-names>Ting-Ting</given-names></name><xref ref-type="aff" rid="aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Liu</surname><given-names>Wen-Juan</given-names></name><xref ref-type="aff" rid="aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Wang</surname><given-names>Xiao-Quan</given-names></name><xref ref-type="aff" rid="aff1"></xref><xref ref-type="corresp" rid="cor1"><sup>*</sup></xref></contrib></contrib-group><aff id="aff1"><addr-line>State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, the Chinese Academy of Sciences, Beijing, China</addr-line></aff><contrib-group><contrib contrib-type="editor"><name><surname>Blazquez</surname><given-names>Miguel A.</given-names></name><role>Editor</role><xref ref-type="aff" rid="edit1"></xref></contrib></contrib-group><aff id="edit1"><addr-line>Instituto de Biología Molecular y Celular de Plantas, Spain</addr-line></aff><author-notes><corresp id="cor1">* E-mail: <email>xiaoq_wang@ibcas.ac.cn</email> (XQW); <email>jinhua_ran@ibcas.ac.cn</email> (JHR)</corresp><fn fn-type="conflict"><p><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p></fn><fn fn-type="con"><p>Conceived and designed the experiments: JHR XQW. Performed the experiments: WJL. Analyzed the data: JHR TTS. Contributed reagents/materials/analysis tools: WJL JHR. Wrote the paper: JHR XQW.</p></fn></author-notes><pub-date pub-type="collection"><year>2013</year></pub-date><pub-date pub-type="epub"><day>11</day><month>11</month><year>2013</year></pub-date><volume>8</volume><issue>11</issue><elocation-id>e78997</elocation-id><history><date date-type="received"><day>9</day><month>8</month><year>2013</year></date><date date-type="accepted"><day>27</day><month>9</month><year>2013</year></date></history><permissions><copyright-year>2013</copyright-year><copyright-holder>Ran et al</copyright-holder><license><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><abstract><p>Stomata play significant roles in plant evolution. A trio of closely related basic Helix-Loop-Helix (bHLH) subgroup Ia genes, <italic>SPCH</italic>, <italic>MUTE</italic> and <italic>FAMA</italic>, mediate sequential steps of stomatal development, and their functions may be conserved in land plants. However, the evolutionary history of the putative <italic>SPCH/MUTE/FAMA</italic> genes is still greatly controversial, especially the phylogenetic positions of the bHLH Ia members from basal land plants. To better understand the evolutionary pattern and functional diversity of the bHLH genes involved in stomatal development, we made a comprehensive evolutionary analysis of the homologous genes from 54 species representing the major lineages of green plants. The phylogenetic analysis indicated: (1) All bHLH Ia genes from the two basal land plants <italic>Physcomitrella</italic> and <italic>Selaginella</italic> were closely related to the <italic>FAMA</italic> genes of seed plants; and (2) the gymnosperm ‘<italic>SPCH</italic>’ genes were sister to a clade comprising the angiosperm <italic>SPCH</italic> and <italic>MUTE</italic> genes, while the <italic>FAMA</italic> genes of gymnosperms and angiosperms had a sister relationship. The revealed phylogenetic relationships are also supported by the distribution of gene structures and previous functional studies. Therefore, we deduce that the function of <italic>FAMA</italic> might be ancestral in the bHLH Ia subgroup. In addition, the gymnosperm “<italic>SPCH”</italic> genes may represent an ancestral state and have a dual function of <italic>SPCH</italic> and <italic>MUTE</italic>, two genes that could have originated from a duplication event in the common ancestor of angiosperms. Moreover, in angiosperms, <italic>SPCHs</italic> have experienced more duplications and harbor more copies than <italic>MUTEs</italic> and <italic>FAMAs</italic>, which, together with variation of the stomatal development in the entry division, implies that <italic>SPCH</italic> might have contributed greatly to the diversity of stomatal development. Based on the above, we proposed a model for the correlation between the evolution of stomatal development and the genes involved in this developmental process in land plants.</p></abstract><funding-group><funding-statement>This work was supported by the National Natural Science Foundation of China (Grant numbers 30870166, 30990240 and 30425028). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.</funding-statement></funding-group><counts><page-count count="11"></page-count></counts></article-meta></front><body><sec id="s1"><title>Introduction</title><p>The origin of terrestrial plants was a key event in the evolutionary history of life on earth <xref ref-type="bibr" rid="pone.0078997-Bateman1">[1]</xref>, <xref ref-type="bibr" rid="pone.0078997-Delaux1">[2]</xref>. During the transition to a terrestrial habitat, ancestral land plants overcame several challenges, including growing in an environment with a limited water and mineral supply, surviving the harmful effects of enhanced ultraviolet and cosmic rays and defending against attack from a new and diversified set of microbes <xref ref-type="bibr" rid="pone.0078997-Delaux1">[2]</xref>. Stomata were a key evolutionary innovation that contributed to overcoming many of these challenges. Stomata first appeared more than 410 million years ago <xref ref-type="bibr" rid="pone.0078997-Edwards1">[3]</xref>, <xref ref-type="bibr" rid="pone.0078997-Raven1">[4]</xref> and generally consist of two guard cells (GCs) surrounding a pore in the epidermis except that a single guard cell that encircles the pore was found in the moss <italic>Funaria</italic><xref ref-type="bibr" rid="pone.0078997-Sack1">[5]</xref>. They play significant roles in protecting plants from a non-aqueous atmosphere by mediating gas exchange integral to photosynthesis and water transportation <xref ref-type="bibr" rid="pone.0078997-Raven1">[4]</xref>, <xref ref-type="bibr" rid="pone.0078997-Torii1">[6]</xref>. Some of the earliest branching land plants, including liverworts, hornworts and some mosses, lack stomata, but all other land plants possess them <xref ref-type="bibr" rid="pone.0078997-Edwards1">[3]</xref>. Fossil evidence shows that the structure of stomata has remained more or less unchanged ever since its origin <xref ref-type="bibr" rid="pone.0078997-Edwards1">[3]</xref>, <xref ref-type="bibr" rid="pone.0078997-Beerling1">[7]</xref>, <xref ref-type="bibr" rid="pone.0078997-Chater1">[8]</xref>, but the developmental processes that lead to stomata have become more complex, such as the occurrence of amplifying divisions, and also more efficient throughout the land plant phylogeny <xref ref-type="bibr" rid="pone.0078997-Raven1">[4]</xref>, <xref ref-type="bibr" rid="pone.0078997-Haworth1">[9]</xref>, <xref ref-type="bibr" rid="pone.0078997-McAdam1">[10]</xref>. These efficiencies in water use, gas exchange and photosynthetic rates are likely key to driving the adaptation of higher plants to a wide array of ecological niches although little is known about possible correlations between stomatal patterning and physiological function <xref ref-type="bibr" rid="pone.0078997-Haworth1">[9]</xref>–<xref ref-type="bibr" rid="pone.0078997-Croxdale1">[19]</xref>.</p><p>To date, most investigations of stomata have focused on their development and their reactions to environmental stressors (e.g., <xref ref-type="bibr" rid="pone.0078997-Edwards1">[3]</xref>, <xref ref-type="bibr" rid="pone.0078997-Haworth1">[9]</xref>, <xref ref-type="bibr" rid="pone.0078997-Brodribb2">[13]</xref>, <xref ref-type="bibr" rid="pone.0078997-Darwin1">[20]</xref>–<xref ref-type="bibr" rid="pone.0078997-McAdam3">[23]</xref>). However, in the last decade some of the genes that regulate stomatal development and patterning have been isolated. In <italic>Arabidopsis</italic>, these genes are a trio of closely related basic Helix-Loop-Helix (bHLH) genes, known as <italic>SPCH</italic> (<italic>SPEECHLESS</italic>), <italic>MUTE</italic>, and <italic>FAMA</italic> (here termed the <italic>SMF</italic> genes). These three genes work together with their heterodimeric partners - <italic>SCRM</italic> (<italic>SCREAM</italic>) and <italic>SCRM2</italic> - to mediate sequential steps of the cell-state transitions that lead to stomatal formation. These steps include: <italic>i</italic>) asymmetric cell division that leads to <italic>ii</italic>) the acquisition of guard mother cell (GMC) identity and, finally, <italic>iii</italic>) the differentiation of guard cells (GCs) <xref ref-type="bibr" rid="pone.0078997-Kanaoka1">[24]</xref>–<xref ref-type="bibr" rid="pone.0078997-Gudesblat1">[28]</xref>.</p><p>The bHLH family, which is typified by the highly conserved bHLH domain, is a large family of transcription factors that is distributed throughout the major eukaryotic lineages <xref ref-type="bibr" rid="pone.0078997-Pires1">[29]</xref>. Phylogenetic analysis indicates that the bHLH genes from <italic>Arabidopsis</italic> are divided into 26 subgroups <xref ref-type="bibr" rid="pone.0078997-OhashiIto1">[27]</xref>, <xref ref-type="bibr" rid="pone.0078997-Pires1">[29]</xref>, <xref ref-type="bibr" rid="pone.0078997-Heim1">[30]</xref>. The three genes that contribute to stomatal development - i.e., <italic>SPCH</italic>, <italic>MUTE</italic>, and <italic>FAMA</italic> - belong to bHLH subgroup Ia, which also includes seven genes of unknown function. In contrast, <italic>SCRM</italic> and <italic>SCRM2</italic> are members of subgroup IIIb <xref ref-type="bibr" rid="pone.0078997-Pires1">[29]</xref>.</p><p>The function of Ia and IIIb genes may be conserved over evolutionary time. For example, functional analyses have shown that PpSMF1 and PpSMF2, two bHLH group Ia members from the bryophyte <italic>Physcomitrella patens</italic>, can recapitulate elements of the <italic>SPCH</italic>, <italic>MUTE</italic> and <italic>FAMA</italic> overexpression phenotypes of <italic>Arabidopsis thaliana</italic><xref ref-type="bibr" rid="pone.0078997-MacAlister2">[31]</xref>. PpSMF1 can also partially complement <italic>mute</italic> and <italic>fama</italic> mutants in <italic>A. thaliana</italic><xref ref-type="bibr" rid="pone.0078997-MacAlister2">[31]</xref>. In addition, the partnership between the subgroup Ia SPCH/MUTE/FAMA and the subgroup IIIb SCRM/SCRM2 might have an ancient origin, because both of these two subgroups occur in the basal land plant, <italic>P. patens</italic><xref ref-type="bibr" rid="pone.0078997-Vaten1">[32]</xref>.</p><p>The evolutionary history of <italic>SPCH/MUTE/FAMA</italic> genes is nonetheless controversial <xref ref-type="bibr" rid="pone.0078997-MacAlister2">[31]</xref>, <xref ref-type="bibr" rid="pone.0078997-Peterson1">[33]</xref> because there is conflicting information about their origins and the relationships of their paralogs. At least two analyses found that the <italic>SPCH</italic> and <italic>MUTE</italic> genes are closest paralogs <xref ref-type="bibr" rid="pone.0078997-Pires1">[29]</xref>, <xref ref-type="bibr" rid="pone.0078997-ToledoOrtiz1">[34]</xref>, <xref ref-type="bibr" rid="pone.0078997-Li1">[35]</xref>, whereas others suggest that <italic>SPCH</italic> and <italic>FAMA</italic> genes are closest paralogs <xref ref-type="bibr" rid="pone.0078997-MacAlister2">[31]</xref>, <xref ref-type="bibr" rid="pone.0078997-Peterson1">[33]</xref>. There are also discrepancies as to whether the homologs from basal land plants fall within the <italic>SPCH</italic>, <italic>MUTE</italic> and <italic>FAMA</italic> clades or represent sister-lineages to these clades <xref ref-type="bibr" rid="pone.0078997-MacAlister2">[31]</xref>. It is important to note that the resolution of these phylogenetic issues will yield insight into the evolution of stomata, because each of the bHLH Ia genes performs a defined role in stomatal development.</p><p>Here we investigate the distribution and evolutionary relationships of bHLH Ia genes among land plants to better understand the evolution of genes involved in stomatal development. Thus far, evolutionary analyses of the members of Ia subgroup have been based on a small sample (n <12) of angiosperm species; here we survey a total of 51 species of land plants (and one green algae and two multicellular algae species) for the presence and distribution of subgroup Ia genes. Based on both phylogenetic and structural analyses of the bHLH Ia genes, we interpret their evolution in land plants, and we also predict a model for the correlation between the evolution of stomatal development and the genes involved in their development.</p></sec><sec sec-type="materials|methods" id="s2"><title>Materials and Methods</title><sec id="s2a"><title>Ethics Statement</title><p>No specific permits were required for the sampling.</p></sec><sec id="s2b"><title>Identification of bHLH Ia Homologs</title><p>We surveyed a number of plant databases – such as Phytozome, NCBI, PGDD, PlantTFDB, EST, SRA and FLcDNA databases and other genome databases (<xref ref-type="table" rid="pone-0078997-t001">Tables 1</xref>, <xref ref-type="table" rid="pone-0078997-t002">2</xref>, <xref ref-type="supplementary-material" rid="pone.0078997.s003">S1</xref>) - to identify bHLH Ia homologs from plant species. To retrieve Ia homologs from these databases, we performed tBLASTn and BLAST+ (ncbi-blast-2.2.27+) searches using the <italic>A. thaliana FAMA</italic> [Phytozome:AT3G24140] amino acid sequence as a query.</p><table-wrap id="pone-0078997-t001" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0078997.t001</object-id><label>Table 1</label><caption><title>Information of the bHLH Ia genes in the sampled plant species with whole genome sequences.</title></caption><alternatives><graphic id="pone-0078997-t001-1" xlink:href="pone.0078997.t001"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col></colgroup><thead><tr><td align="left" rowspan="1" colspan="1">Species</td><td align="left" rowspan="1" colspan="1">Abbr.</td><td colspan="4" align="left" rowspan="1">Copy number</td><td align="left" rowspan="1" colspan="1">Database*</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"><italic>FAMA</italic></td><td align="left" rowspan="1" colspan="1"><italic>MUTE</italic></td><td align="left" rowspan="1" colspan="1"><italic>SPCH</italic></td><td align="left" rowspan="1" colspan="1">Other Ias</td><td align="left" rowspan="1" colspan="1"></td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1"><italic>Volvox carteri</italic></td><td align="left" rowspan="1" colspan="1">Vca</td><td align="left" rowspan="1" colspan="1">0</td><td align="left" rowspan="1" colspan="1">0</td><td align="left" rowspan="1" colspan="1">0</td><td align="left" rowspan="1" colspan="1">0</td><td align="left" rowspan="1" colspan="1">Phytozome</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Physcomitrella patens</italic></td><td align="left" rowspan="1" colspan="1">Ppa</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">0</td><td align="left" rowspan="1" colspan="1">0</td><td align="left" rowspan="1" colspan="1">0</td><td align="left" rowspan="1" colspan="1">Phytozome/PGDD</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Selaginella moellendorffii</italic></td><td align="left" rowspan="1" colspan="1">Smo</td><td align="left" rowspan="1" colspan="1">3</td><td align="left" rowspan="1" colspan="1">0</td><td align="left" rowspan="1" colspan="1">0</td><td align="left" rowspan="1" colspan="1">0</td><td align="left" rowspan="1" colspan="1">Phytozome/PGDD</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Amborella trichopoda</italic></td><td align="left" rowspan="1" colspan="1">Atr</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">Amborella GD</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Aquilegia coerulea</italic></td><td align="left" rowspan="1" colspan="1">Aco</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">Phytozome</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Arabidopsis lyrata</italic></td><td align="left" rowspan="1" colspan="1">Aly</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">7</td><td align="left" rowspan="1" colspan="1">Phytozome/PGDD</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Arabidopsis thaliana</italic></td><td align="left" rowspan="1" colspan="1">Ath</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">7</td><td align="left" rowspan="1" colspan="1">Phytozome/PGDD</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Brachypodium distachyon</italic></td><td align="left" rowspan="1" colspan="1">Bdi</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">7</td><td align="left" rowspan="1" colspan="1">Phytozome/PGDD</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Brassica rapa</italic></td><td align="left" rowspan="1" colspan="1">Bra</td><td align="left" rowspan="1" colspan="1">3</td><td align="left" rowspan="1" colspan="1">3</td><td align="left" rowspan="1" colspan="1">3</td><td align="left" rowspan="1" colspan="1">14</td><td align="left" rowspan="1" colspan="1">Phytozome/PGDD</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Cajanus cajan</italic></td><td align="left" rowspan="1" colspan="1">Cca</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">7</td><td align="left" rowspan="1" colspan="1">IIPG/PGDD</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Capsella rubella</italic></td><td align="left" rowspan="1" colspan="1">Cru</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">9</td><td align="left" rowspan="1" colspan="1">Phytozome</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Carica papaya</italic></td><td align="left" rowspan="1" colspan="1">Cpa</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">0</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">4</td><td align="left" rowspan="1" colspan="1">Phytozome/PGDD</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Citrus clementina</italic></td><td align="left" rowspan="1" colspan="1">Ccl</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">3</td><td align="left" rowspan="1" colspan="1">Phytozome</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Citrus sinensis</italic></td><td align="left" rowspan="1" colspan="1">Csi</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">0</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">4</td><td align="left" rowspan="1" colspan="1">Phytozome</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Cucumis sativus</italic></td><td align="left" rowspan="1" colspan="1">Csa</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">5</td><td align="left" rowspan="1" colspan="1">Phytozome/PGDD</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Eucalyptus grandis</italic></td><td align="left" rowspan="1" colspan="1">Egr</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">5</td><td align="left" rowspan="1" colspan="1">Phytozome</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Fragaria vesca</italic></td><td align="left" rowspan="1" colspan="1">Fve</td><td align="left" rowspan="1" colspan="1">0</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">0</td><td align="left" rowspan="1" colspan="1">6</td><td align="left" rowspan="1" colspan="1">PFR/PGDD</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Glycine max</italic></td><td align="left" rowspan="1" colspan="1">Gma</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">4</td><td align="left" rowspan="1" colspan="1">12</td><td align="left" rowspan="1" colspan="1">Phytozome/PGDD</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Linum usitatissimum</italic></td><td align="left" rowspan="1" colspan="1">Lus</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">4</td><td align="left" rowspan="1" colspan="1">7</td><td align="left" rowspan="1" colspan="1">Phytozome</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Lotus japonicus</italic></td><td align="left" rowspan="1" colspan="1">Lja</td><td align="left" rowspan="1" colspan="1">0</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">0</td><td align="left" rowspan="1" colspan="1">6</td><td align="left" rowspan="1" colspan="1">Kazusa/PGDD</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Malus domestica</italic></td><td align="left" rowspan="1" colspan="1">Mdo</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">8</td><td align="left" rowspan="1" colspan="1">Phytozome/PGDD</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Manihot esculenta</italic></td><td align="left" rowspan="1" colspan="1">Mes</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">7</td><td align="left" rowspan="1" colspan="1">Phytozome</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Medicago truncatula</italic></td><td align="left" rowspan="1" colspan="1">Mtr</td><td align="left" rowspan="1" colspan="1">0</td><td align="left" rowspan="1" colspan="1">0</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">5</td><td align="left" rowspan="1" colspan="1">Phytozome/PGDD</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Mimulus guttatus</italic></td><td align="left" rowspan="1" colspan="1">Mgu</td><td align="left" rowspan="1" colspan="1">0</td><td align="left" rowspan="1" colspan="1">0</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">Phytozome</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Musa acuminata</italic></td><td align="left" rowspan="1" colspan="1">Mac</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">3</td><td align="left" rowspan="1" colspan="1">16</td><td align="left" rowspan="1" colspan="1">Banana Genome/PGDD</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Oryza sativa</italic></td><td align="left" rowspan="1" colspan="1">Osa</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">8</td><td align="left" rowspan="1" colspan="1">Phytozome/PGDD</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Phaseolus vulgaris</italic></td><td align="left" rowspan="1" colspan="1">Pvu</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">6</td><td align="left" rowspan="1" colspan="1">Phytozome</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Phoenix dactylifera</italic></td><td align="left" rowspan="1" colspan="1">Pda</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">5</td><td align="left" rowspan="1" colspan="1">Date Palm Draft Sequence</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Populus trichocarpa</italic></td><td align="left" rowspan="1" colspan="1">Ptr</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">9</td><td align="left" rowspan="1" colspan="1">Phytozome/PGDD</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Prunus persica</italic></td><td align="left" rowspan="1" colspan="1">Per</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">5</td><td align="left" rowspan="1" colspan="1">Phytozome/PGDD</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Ricinus communis</italic></td><td align="left" rowspan="1" colspan="1">Rco</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">4</td><td align="left" rowspan="1" colspan="1">Phytozome/PGDD</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Setaria italica</italic></td><td align="left" rowspan="1" colspan="1">Sit</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">8</td><td align="left" rowspan="1" colspan="1">Phytozome</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Solanum lycopersicum</italic></td><td align="left" rowspan="1" colspan="1">Sly</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">0</td><td align="left" rowspan="1" colspan="1">4</td><td align="left" rowspan="1" colspan="1">SGN/PGDD</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Solanum tuberosum</italic></td><td align="left" rowspan="1" colspan="1">Stu</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">0</td><td align="left" rowspan="1" colspan="1">0</td><td align="left" rowspan="1" colspan="1">4</td><td align="left" rowspan="1" colspan="1">PGSC/PGDD</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Sorghum bicolor</italic></td><td align="left" rowspan="1" colspan="1">Sbi</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">7</td><td align="left" rowspan="1" colspan="1">Phytozome/PGDD</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Thellungiella halophila</italic></td><td align="left" rowspan="1" colspan="1">Tha</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">8</td><td align="left" rowspan="1" colspan="1">Phytozome</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Theobroma cacao</italic></td><td align="left" rowspan="1" colspan="1">Tca</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">5</td><td align="left" rowspan="1" colspan="1">CIRAD/PGDD</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Vitis vinifera</italic></td><td align="left" rowspan="1" colspan="1">Vvi</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">5</td><td align="left" rowspan="1" colspan="1">Phytozome/PGDD</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Zea mays</italic></td><td align="left" rowspan="1" colspan="1">Zma</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">3</td><td align="left" rowspan="1" colspan="1">10</td><td align="left" rowspan="1" colspan="1">Phytozome/PGDD</td></tr></tbody></table></alternatives><table-wrap-foot><fn id="nt101"><label></label><p>Abbr., Abbreviation.*, database websites: Amborella GD, <ext-link ext-link-type="uri" xlink:href="http://www.amborella.org/">http://www.amborella.org/</ext-link>; Banana Genome, <ext-link ext-link-type="uri" xlink:href="http://banana-genome.cirad.fr/">http://banana-genome.cirad.fr/</ext-link>; CIRAD, <ext-link ext-link-type="uri" xlink:href="http://cocoagendb.cirad.fr/gbrowse/download.html">http://cocoagendb.cirad.fr/gbrowse/download.html</ext-link>; Date Palm Draft Sequence, <ext-link ext-link-type="uri" xlink:href="http://qatar-weill.cornell.edu/research/datepalmGenome/download.html">http://qatar-weill.cornell.edu/research/datepalmGenome/download.html</ext-link>; IIPG, <ext-link ext-link-type="uri" xlink:href="http://www.icrisat.org/gt-bt/iipg/Home.html">http://www.icrisat.org/gt-bt/iipg/Home.html</ext-link>; Kazusa, <ext-link ext-link-type="ftp" xlink:href="ftp://ftp.kazusa.or.jp/pub/lotus/">ftp://ftp.kazusa.or.jp/pub/lotus/</ext-link>; NCBI, <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/">http://www.ncbi.nlm.nih.gov/</ext-link>; Phytozome, <ext-link ext-link-type="uri" xlink:href="http://www.phytozome.net">http://www.phytozome.net</ext-link>; PFR, <ext-link ext-link-type="uri" xlink:href="http://www.strawberrygenome.org/">http://www.strawberrygenome.org/</ext-link>; PGSC, <ext-link ext-link-type="uri" xlink:href="http://potatogenomics.plantbiology.msu.edu">http://potatogenomics.plantbiology.msu.edu</ext-link>; PlantTFDB, <ext-link ext-link-type="uri" xlink:href="http://planttfdb.cbi.edu.cn/">http://planttfdb.cbi.edu.cn/</ext-link>.</p></fn></table-wrap-foot></table-wrap><table-wrap id="pone-0078997-t002" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0078997.t002</object-id><label>Table 2</label><caption><title>Information of the bHLH Ia genes in the plant species with EST or SRA databases.</title></caption><alternatives><graphic id="pone-0078997-t002-2" xlink:href="pone.0078997.t002"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col></colgroup><thead><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">Species</td><td colspan="4" align="left" rowspan="1">Copy Number</td><td align="left" rowspan="1" colspan="1">EST & cDNA</td><td align="left" rowspan="1" colspan="1">SRA (spots)</td><td align="left" rowspan="1" colspan="1">SRA Submission</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"><italic>SPCH</italic></td><td align="left" rowspan="1" colspan="1"><italic>MUTE</italic></td><td align="left" rowspan="1" colspan="1"><italic>FAMA</italic></td><td align="left" rowspan="1" colspan="1">Other Ias</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1"><bold>Green algae</bold></td><td align="left" rowspan="1" colspan="1"><italic>Nitella hyalina</italic></td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">88,280</td><td align="left" rowspan="1" colspan="1">949,065</td><td align="left" rowspan="1" colspan="1">SRA023590</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"><italic>Nitella mirabilis</italic></td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">83,526</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Liverwort</bold></td><td align="left" rowspan="1" colspan="1"><italic>Marchantia polymorpha</italic></td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">33,722</td><td align="left" rowspan="1" colspan="1">22,854,396</td><td align="left" rowspan="1" colspan="1">SRA026315</td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Fern</bold></td><td align="left" rowspan="1" colspan="1"><italic>Adiantum capillus-veneris</italic></td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">30,544</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Gymnosperms</bold></td><td align="left" rowspan="1" colspan="1"><italic>Cephalotaxus harringtonia</italic></td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">695,559</td><td align="left" rowspan="1" colspan="1">SRA023613</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"><italic>Ginkgo biloba</italic></td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">3</td><td align="left" rowspan="1" colspan="1">21,709</td><td align="left" rowspan="1" colspan="1">64,057</td><td align="left" rowspan="1" colspan="1">SRA030487</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"><italic>Gnetum gnemon</italic></td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">10,756</td><td align="left" rowspan="1" colspan="1">432,517</td><td align="left" rowspan="1" colspan="1">SRA023615</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"><italic>Picea glauca#</italic></td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">321,713</td><td align="left" rowspan="1" colspan="1">10,922,903</td><td align="left" rowspan="1" colspan="1">SRA023921</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"><italic>Picea breweriana*</italic></td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"><italic>Picea jezoensis*</italic></td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"><italic>Picea smithiana*</italic></td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"><italic>Picea sitchensis</italic></td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">206,402</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"><italic>Pinus banksiana</italic></td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">36,387</td><td align="left" rowspan="1" colspan="1">1,397,993</td><td align="left" rowspan="1" colspan="1">SRA048732</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"><italic>Pinus taeda</italic></td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">7</td><td align="left" rowspan="1" colspan="1">329,066</td><td align="left" rowspan="1" colspan="1">4,331,325</td><td align="left" rowspan="1" colspan="1">SRA023533</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"><italic>Sciadopitys verticilliata</italic></td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">–</td><td align="left" rowspan="1" colspan="1">484,806</td><td align="left" rowspan="1" colspan="1">SRA023758</td></tr></tbody></table></alternatives><table-wrap-foot><fn id="nt102"><label></label><p>#, Sequences from FLcDNA database of Arborea; *, sequences from PCR amplification; -, missing information.</p></fn></table-wrap-foot></table-wrap><p>We subjected the identified sequences to three additional filters. First, we discarded sequences that had lower sequence similarities to AT3G24140 than did the first non-subgroup-Ia member from <italic>A. thaliana</italic> [Phytozome: AT2G22750, a member of IVa]. Second, we examined individual sequences for two characteristic features of subgroup Ia genes: the bHLH domain and the SMF domain <xref ref-type="bibr" rid="pone.0078997-Heim1">[30]</xref>, <xref ref-type="bibr" rid="pone.0078997-MacAlister2">[31]</xref>. These two domains were identified by Heim <italic>et al.</italic><xref ref-type="bibr" rid="pone.0078997-Heim1">[30]</xref>. Any sequence that lacked one of the domains was removed from further analysis. In addition, 36 putative Ia genes were modified by hand for a better alignment with the bHLH or SMF domain (<xref ref-type="supplementary-material" rid="pone.0078997.s003">Table S1</xref>). Finally, we culled redundant transcripts, sequences with incomplete bHLH or sequences with unalignable C-terminal domains from the final dataset.</p></sec><sec id="s2c"><title>DNA and RNA Extraction, PCR and RT-PCR Amplification, Cloning and Sequencing</title><p>Two types of bHLH Ia genes that grouped with <italic>SPCH/MUTE/FAMA</italic> genes were found in eight gymnosperm species, but we found no species that had both types. To assess whether these two types existed in one species, we amplified these Ia genes from the DNA and cDNA of three <italic>Picea</italic> species (<italic>Picea breweriana</italic>, <italic>P. jezoensis</italic>, and <italic>P. smithiana</italic>). Genomic DNAs were extracted from leaves using the modified cetyltrimethylammonium bromide (CTAB) method <xref ref-type="bibr" rid="pone.0078997-Doyle1">[36]</xref>, <xref ref-type="bibr" rid="pone.0078997-Rogers1">[37]</xref>. Total RNA extraction, purification and first-strand cDNA synthesis followed the protocols of Guo <italic>et al</italic>. <xref ref-type="bibr" rid="pone.0078997-Guo1">[38]</xref>. The primers used to amplify the Ia genes were designed to the EST sequence [GenBank:GW768467] of <italic>Pinus banksiana</italic> and the predicted unigene [Arborea:GQ04006] from <italic>Picea glauca</italic>, including FAMA-U5F3 and FAMA-U3R as well as bHLH2-U5F and bHLH2-U3R1 (<xref ref-type="supplementary-material" rid="pone.0078997.s004">Table S2</xref>). Polymerase chain reaction (PCR), product purification, cloning and sequencing followed the protocols of Ran <italic>et al</italic>. <xref ref-type="bibr" rid="pone.0078997-Ran1">[39]</xref> except an annealing and sequencing reaction temperature of 60°C. In addition, some internal primers were used in sequencing (<xref ref-type="supplementary-material" rid="pone.0078997.s004">Table S2</xref>).</p></sec><sec id="s2d"><title>Phylogenetic Reconstruction</title><p>Coding sequences of bHLH Ia genes were aligned using the program Clustal X version 2.0 <xref ref-type="bibr" rid="pone.0078997-Thompson1">[40]</xref>. The conserved bHLH and SMF domains (a total of 468 bp or 156 aa) <xref ref-type="bibr" rid="pone.0078997-MacAlister2">[31]</xref> were used for the phylogenetic analyses. Substitution saturation was tested using DAMBE 5.3.00 <xref ref-type="bibr" rid="pone.0078997-Xia1">[41]</xref>, which showed that the third positions were saturated. jModeltest 2 <xref ref-type="bibr" rid="pone.0078997-Darriba1">[42]</xref> and ProtTest 3.2 <xref ref-type="bibr" rid="pone.0078997-Darriba2">[43]</xref> were used to identify the best-fit models of nucleotide and amino acid (AA) sequence evolution, respectively. Using both Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC), the GTR+I+G model was selected for nucleotide sequence analyses and the JTT+I+G model for AA analyses. Bayesian and maximum-likelihood (ML) trees were constructed with MrBayes version 3.1.2 <xref ref-type="bibr" rid="pone.0078997-Huelsenbeck1">[44]</xref> and PhyML version 2.4.4 <xref ref-type="bibr" rid="pone.0078997-Guindon1">[45]</xref>, respectively.</p></sec><sec id="s2e"><title>Selection Test and Nucleotide Diversity Analysis</title><p>ML analyses were performed to identify regions or clades of the bHLH Ia genes that may have been subject to diversifying selection, using the Fitmodel program version 0.5.3 <xref ref-type="bibr" rid="pone.0078997-Guindon2">[46]</xref> and the codeml program of PAML version 4.6 <xref ref-type="bibr" rid="pone.0078997-Yang1">[47]</xref>, respectively. Because the Ia gene dataset was very large, we limited these analyses to a subset of subgroup Ia sequences. <italic>SPCH/MUTE/FAMA</italic> homologs, all bHLH other Ias from gymnosperms, all bHLH Ias from <italic>Physcomitrella</italic> and <italic>Selaginella</italic>, and some of bHLH other Ias from four angiosperm species (<italic>Arabidopsis thaliana</italic>, <italic>Glycine max</italic>, <italic>Linum usitatissimum</italic> and <italic>Oryza sativa</italic>) were analyzed with Fitmodel, in which the M0, M3, M3+S1 and M3+S2 models were applied. Three genic classes, including clade A, and Ang-MUTE and Ang-SPCH in clade B (<xref ref-type="fig" rid="pone-0078997-g001">Fig. 1A</xref>), were analyzed with the branch-site and site models (codeml program), respectively. The parameter settings followed Guo <italic>et al</italic>. <xref ref-type="bibr" rid="pone.0078997-Guo1">[38]</xref>. To test the evolutionary rate variation, DnaSP version 5.10.00 <xref ref-type="bibr" rid="pone.0078997-Librado1">[48]</xref> was used to calculate the nucleotide diversity (<italic>Pi</italic>), the number of nonsynonmous substitutions per nonsynonymous site (<italic>Ka</italic>), the number of synonymous substitutions per synonymous site (<italic>Ks</italic>), and the ω (<italic>Ka</italic>/<italic>Ks</italic>) value for the sequences of the angiosperm <italic>FAMA</italic>, <italic>MUTE</italic>, <italic>SPCH</italic> and other Ia genes (<xref ref-type="fig" rid="pone-0078997-g001">Fig. 1</xref>) because some clades lack members from non-flowering plants.</p><fig id="pone-0078997-g001" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0078997.g001</object-id><label>Figure 1</label><caption><title>Maximum likelihood (ML) trees of the bHLH Ia genes constructed based on the nucleotide sequences.</title><p>A. The tree was rooted with the Ia genes from <italic>Physcomitrella patens</italic>; B. The tree was not rooted. Numbers above branches indicate bootstrap values higher than 50%. Ang, angiosperm; Gym, gymnosperm; Smo, <italic>Selaginella moellendorffii</italic>; Ppa, <italic>Physcomitrella patens</italic>.</p></caption><graphic xlink:href="pone.0078997.g001"></graphic></fig></sec></sec><sec id="s3"><title>Results</title><sec id="s3a"><title>Sequence Characterization</title><p>We searched complete genomes as well as EST and SRA resources to identify bHLH Ia homologs in basal (non-angiosperm) land plants. Using Blast-based strategies (see Materials and Methods) with a loose searching criteria (E value = 1), we characterized two and three SMF genes in the bryophyte <italic>Physcomitrella patens</italic> and the lycopsid <italic>Selaginella moellendorffii</italic>, respectively. However, using similar search methods, we were unable to identify bHLH Ia homologs from the complete genome of a green algae (<italic>Volvox carteri</italic>), and the EST+cDNA databases of two multicellular green alga (<italic>Nitella hyalina</italic> and <italic>Nitella mirabilis</italic>) (88,280 and 88,526 sequences, respectively), a liverwort (<italic>Marchantia polymorpha</italic>) (33722 sequences) and a fern <italic>Adiatum capillus-veneris</italic> (30,544 sequences) (<xref ref-type="table" rid="pone-0078997-t002">Table 2</xref>). The inability to identify Ia homologs from EST databases of the liverwort and fern could reflect reality, or it could be caused by the size of EST databases. We note, however, that other non-Ia bHLH genes were successfully retrieved from these species. In addition, we identified 19 Ia homologs from the EST or SRA databases of eight gymnosperms (<xref ref-type="supplementary-material" rid="pone.0078997.s003">Table S1</xref>), and PCR-amplified six sequences from three spruce species (<xref ref-type="table" rid="pone-0078997-t002">Table 2</xref>). Of the 25 sequences from gymnosperms, 4, 6 and 15 showed similar gene structures with <italic>SPCH</italic>, <italic>FAMA</italic> and Other Ias, respectively.</p><p>Within angiosperms, SMF (<italic>SPCH</italic>, <italic>MUTE</italic> and <italic>FAMA</italic>) homologs were identified in all 36 species sampled, but homologs of individual genes were not found in five species for <italic>FAMA</italic>, four species for <italic>SPCH</italic> and five species for <italic>MUTE</italic>. However, 14, 6, and 8 species harbored more than one <italic>SPCH</italic>, <italic>MUTE</italic> and <italic>FAMA</italic> homolog (<xref ref-type="table" rid="pone-0078997-t001">Table 1</xref>). In addition, 2–16 copies of other (i.e., non-SPCH, non-MUTE and non-FAMA) Ia members were found throughout the 36 angiosperm species. In total, 395 bHLH Ia sequences from 49 species of land plants were used in the final analyses (<xref ref-type="supplementary-material" rid="pone.0078997.s003">Table S1</xref>). These coding sequences ranged from 543 (AcoMUTE) to 3114 bp (FveIa_1).</p><p>All of the sequences in the final dataset contained both bHLH and SMF domains, but additional domains were also detected (<xref ref-type="fig" rid="pone-0078997-g002">Fig. 2</xref>), as suggested by MacAlister and Bergmann <xref ref-type="bibr" rid="pone.0078997-MacAlister2">[31]</xref>. These domains helped to differentiate gene structure among putative <italic>SPCH</italic>, <italic>MUTE</italic> and <italic>FAMA</italic> genes and also to corroborate phylogenetic inferences based on the bHLH and SMF domain sequences (see below). In angiosperms, the <italic>FAMA</italic> homologs have a pre-bHLH domain (II) and two unique and highly conserved regions; one of these (I) is in the N-terminus between the start codon and the bHLH domain and the other (IV) is immediately N-terminal to the SMF domain. However, four putative <italic>FAMA</italic> homologs (AtrFAMA CpaFAMA, EgrFAMA, and StuFAMA) lacked region I. All <italic>MUTE</italic> homologs lacked the pre-bHLH domain (II) and had a unique pre-SMF domain (V). Finally, all <italic>SPCH</italic> homologs had a MPKTD domain, but it differed in length.</p><fig id="pone-0078997-g002" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0078997.g002</object-id><label>Figure 2</label><caption><title>Structural diagram of the bHLH Ia genes from some representative species of land plants.</title><p>The regions that are unique to <italic>FAMA</italic>, <italic>MUTE</italic>, and <italic>SPCH</italic> are marked with different colors.</p></caption><graphic xlink:href="pone.0078997.g002"></graphic></fig><p>The gene structures in non-angiosperm taxa merit special attention. In gymnosperms, two structures were found, one similar to angiosperm <italic>FAMA</italic> genes and the other similar to angiosperm <italic>SPCH</italic> genes. In <italic>Selaginella moellendorffii</italic>, the three bHLH Ia genes are structurally similar to the angiosperm <italic>FAMA</italic> genes, especially in domains II and IV, and the domain I was also found in SmoSMF1 and SmoSMF2. In <italic>Physcomitrella patens</italic>, the two Ia genes are also similar to the angiosperm <italic>FAMA</italic> genes due to the presence of domains II and IV. Moreover, the other Ia genes also have domain II (<xref ref-type="fig" rid="pone-0078997-g002">Fig. 2</xref>). In brief, according to the gene structure analysis, the basal land plants (bryophyte and lycopsid) only harbored <italic>FAMA</italic> Ia genes, while both <italic>FAMA</italic> and <italic>SPCH</italic> Ia genes occurred in gymnosperms. This was consistent with the reconstruction of ancestral gene structure using Mesquite version 2.75 <xref ref-type="bibr" rid="pone.0078997-Maddison1">[49]</xref>, which is shown in <xref ref-type="supplementary-material" rid="pone.0078997.s001">Fig. S1</xref>. The conserved sequence patterns of the angiosperm <italic>SPCH</italic> and <italic>MUTE</italic> genes as well as the gymnosperm ‘<italic>SPCH</italic>’ genes were generated with WebLogo3 (<ext-link ext-link-type="uri" xlink:href="http://weblogo.berkeley.edu/">http://weblogo.berkeley.edu/</ext-link>) and shown in <xref ref-type="fig" rid="pone-0078997-g003">Fig. 3</xref>.</p><fig id="pone-0078997-g003" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0078997.g003</object-id><label>Figure 3</label><caption><title>Graphical sequence logo representation of bHLH and SMF domains of seed-plant <italic>SPCH/MUTE</italic> genes.</title><p>The conserved sequence pattern was generated using WebLogo3 (<ext-link ext-link-type="uri" xlink:href="http://weblogo.berkeley.edu/">http://weblogo.berkeley.edu/</ext-link>). Bits represent the relative frequency of amino acids.</p></caption><graphic xlink:href="pone.0078997.g003"></graphic></fig></sec><sec id="s3b"><title>Phylogenetic Analyses</title><p>The ML trees generated by nucleotide and AA sequences of the bHLH Ia genes are topologically identical except some branches with low bootstrap support (<xref ref-type="fig" rid="pone-0078997-g001">Figs. 1</xref>, <xref ref-type="supplementary-material" rid="pone.0078997.s002">S2</xref>). Because most Bayesian prosterior probabilities were lower than 0.90, we did not consider the Bayesian tree. The ML trees of the nucleotide sequences with and without outgroups are shown in <xref ref-type="fig" rid="pone-0078997-g001">Figure 1A and 1B</xref>, respectively. From the rooted trees, we found that the Ia genes of vascular plants are monophyletic with high bootstrap support (100%) and could be divided into three main clades (A, B and C) (<xref ref-type="fig" rid="pone-0078997-g001">Figs. 1A</xref>, <xref ref-type="supplementary-material" rid="pone.0078997.s002">S2A, S2B</xref>). Clade A included the SMF genes from <italic>Selaginella moellendorffii</italic> and the <italic>FAMA</italic> homologs from seed plants (gymnosperm and angiosperm). Clade B comprised two sister subclades, i.e., the gymnosperm ‘<italic>SPCH</italic>’ genes and the angiosperm <italic>MUTE</italic> and <italic>SPCH</italic> genes. Clade C comprised all the other Ia genes from gymnosperms and angiosperms. From the unrooted tree, the bHLH Ia members from <italic>Physcomitrella patens</italic> grouped with Clade A (<xref ref-type="fig" rid="pone-0078997-g001">Figs. 1B</xref>, <xref ref-type="supplementary-material" rid="pone.0078997.s002">S2C</xref>).</p></sec><sec id="s3c"><title>Selection Test and Nucleotide Diversity</title><p>The likelihood ratio test (LRT) of the nested models in the Fitmodel program showed that the M3+S1 model was significantly better than the other ones. In the M3+S1 model, the switching rates between ω values (ω1 to ω2, ω1 to ω3, and ω2 to ω3) are equally imposed. Under this model, no codon or branch was inferred to be under relaxed selection (ω3 = 0.32) (<xref ref-type="table" rid="pone-0078997-t003">Table 3</xref>).</p><table-wrap id="pone-0078997-t003" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0078997.t003</object-id><label>Table 3</label><caption><title>Results of the LRT test of the models in Fitmodel for the bHLH Ia genes.</title></caption><alternatives><graphic id="pone-0078997-t003-3" xlink:href="pone.0078997.t003"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col></colgroup><thead><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">M0</td><td align="left" rowspan="1" colspan="1">M3</td><td align="left" rowspan="1" colspan="1">M3+S1</td><td align="left" rowspan="1" colspan="1">M3+S2</td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1"><bold><italic>lnL</italic></bold></td><td align="left" rowspan="1" colspan="1">−26059.06</td><td align="left" rowspan="1" colspan="1">−25848.39</td><td align="left" rowspan="1" colspan="1">−25656.58</td><td align="left" rowspan="1" colspan="1">−25707.45</td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>ω1 ω2 ω3</bold></td><td align="left" rowspan="1" colspan="1">0.08</td><td align="left" rowspan="1" colspan="1">0.01 0.07 0.20</td><td align="left" rowspan="1" colspan="1">0.00 0.07 0.32</td><td align="left" rowspan="1" colspan="1">0.01 0.19 58.10</td></tr><tr><td align="left" rowspan="1" colspan="1"><bold><italic>p</italic></bold><bold>1 </bold><bold><italic>p</italic></bold><bold>2 </bold><bold><italic>p</italic></bold><bold>3</bold></td><td align="left" rowspan="1" colspan="1">1.00</td><td align="left" rowspan="1" colspan="1">0.31 0.51 0.17</td><td align="left" rowspan="1" colspan="1">0.40 0.42 0.18</td><td align="left" rowspan="1" colspan="1">0.65 0.34 0.00</td></tr><tr><td align="left" rowspan="1" colspan="1"><bold><italic>R</italic></bold><bold>01 </bold><bold><italic>R</italic></bold><bold>02 </bold><bold><italic>R</italic></bold><bold>12</bold></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">1.59 1.59 1.59</td><td align="left" rowspan="1" colspan="1">1.66 9.02 434.75</td></tr></tbody></table></alternatives></table-wrap><p>The site model test indicated that no site in the three classes A, Ang-MUTE and Ang-SPCH (<xref ref-type="fig" rid="pone-0078997-g001">Fig. 1</xref>) experienced positive selection. Using the branch-site model, no positively selected site was found in the angiosperm <italic>SPCH</italic> or <italic>MUTE</italic> genes when they acted as the background clade to each other. For the <italic>FAMA</italic> homologs, four branches (vascular plants, seed plants, angiosperms, and eudicots) were tested, and one positively selected site was detected in the branches of seed plants and angiosperms, respectively, but the positive selection was not supported by the LRT test due to a significance much higher than 0.05 (<xref ref-type="supplementary-material" rid="pone.0078997.s005">Table S3</xref>).</p><p>For angiosperms, the <italic>FAMA</italic> genes showed the lowest <italic>Pi</italic>, <italic>Ka</italic> and <italic>ω</italic> values, while the <italic>SPCH</italic> and <italic>MUTE</italic> genes had similar <italic>Pi</italic>, <italic>Ka</italic>, <italic>Ks</italic> and <italic>ω</italic> values. In addition, the highest <italic>Pi</italic>, <italic>Ka</italic>, <italic>Ks</italic> and <italic>ω</italic> values were found in the other Ia genes (<xref ref-type="table" rid="pone-0078997-t004">Table 4</xref>).</p><table-wrap id="pone-0078997-t004" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0078997.t004</object-id><label>Table 4</label><caption><title>Sequence information of angiosperm <italic>SPCH</italic>, <italic>MUTE</italic>, <italic>FAMA</italic> and Other Ias.</title></caption><alternatives><graphic id="pone-0078997-t004-4" xlink:href="pone.0078997.t004"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col></colgroup><thead><tr><td align="left" rowspan="1" colspan="1">Dataset</td><td align="left" rowspan="1" colspan="1"><italic>N</italic></td><td align="left" rowspan="1" colspan="1"><italic>Pi</italic></td><td align="left" rowspan="1" colspan="1"><italic>Ka</italic></td><td align="left" rowspan="1" colspan="1"><italic>Ks</italic></td><td align="left" rowspan="1" colspan="1"><italic>ω</italic> (<italic>Ka/Ks</italic>)</td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1"><italic>SPCH</italic></td><td align="left" rowspan="1" colspan="1">53</td><td align="left" rowspan="1" colspan="1">0.25742±0.00454</td><td align="left" rowspan="1" colspan="1">0.11749</td><td align="left" rowspan="1" colspan="1">0.69853</td><td align="left" rowspan="1" colspan="1">0.168</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>MUTE</italic></td><td align="left" rowspan="1" colspan="1">38</td><td align="left" rowspan="1" colspan="1">0.25526±0.00887</td><td align="left" rowspan="1" colspan="1">0.11350</td><td align="left" rowspan="1" colspan="1">0.73050</td><td align="left" rowspan="1" colspan="1">0.155</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>FAMA</italic></td><td align="left" rowspan="1" colspan="1">41</td><td align="left" rowspan="1" colspan="1">0.21573±0.00868</td><td align="left" rowspan="1" colspan="1">0.05844</td><td align="left" rowspan="1" colspan="1">0.72591</td><td align="left" rowspan="1" colspan="1">0.091</td></tr><tr><td align="left" rowspan="1" colspan="1">Other Ias</td><td align="left" rowspan="1" colspan="1">233</td><td align="left" rowspan="1" colspan="1">0.29867±0.00363</td><td align="left" rowspan="1" colspan="1">0.16091</td><td align="left" rowspan="1" colspan="1">0.75129</td><td align="left" rowspan="1" colspan="1">0.214</td></tr></tbody></table></alternatives><table-wrap-foot><fn id="nt103"><label></label><p><italic>N</italic>, Number of sequences; <italic>Pi</italic>, Nucleotide diversity; <italic>Ka</italic>, The number of nonsynonymous substitutions per nonsynonymous site; <italic>Ks</italic>: The number of synonymous substitutions per synonymous site; ω: <italic>Ka</italic>/<italic>Ks</italic>.</p></fn></table-wrap-foot></table-wrap></sec></sec><sec id="s4"><title>Discussion</title><sec id="s4a"><title>Evolutionary History of the bHLH Ia Genes Involved in Stomatal Development</title><p>Genome-wide analyses of the bHLH transcription factor family have found that SPCH, MUTE and FAMA, together with some other proteins of unknown functions, form a well supported clade (subgroup Ia) <xref ref-type="bibr" rid="pone.0078997-OhashiIto1">[27]</xref>, <xref ref-type="bibr" rid="pone.0078997-Pires1">[29]</xref>, <xref ref-type="bibr" rid="pone.0078997-Heim1">[30]</xref>, <xref ref-type="bibr" rid="pone.0078997-Li1">[35]</xref>, which is characterized by a conserved region of 18 amino acids immediately N-terminal to the bHLH domain and a domain at the C-terminus of the protein <xref ref-type="bibr" rid="pone.0078997-Heim1">[30]</xref>, <xref ref-type="bibr" rid="pone.0078997-MacAlister2">[31]</xref>. Some previous phylogenetic analyses found that the <italic>SPCH</italic> and <italic>MUTE</italic> genes were the closest sister paralogs (The <italic>SPCH</italic> genes were not monophyletic) and the <italic>FAMA</italic> genes grouped with some other subgroup Ia members <xref ref-type="bibr" rid="pone.0078997-Pires1">[29]</xref>, <xref ref-type="bibr" rid="pone.0078997-ToledoOrtiz1">[34]</xref>, <xref ref-type="bibr" rid="pone.0078997-Li1">[35]</xref>. However, other studies that focused on the SMF (<italic>SPCH</italic>, <italic>MUTE</italic>, <italic>FAMA</italic>) genes did not obtain the same phylogenetic relationships. For example, Liu <italic>et al</italic>. <xref ref-type="bibr" rid="pone.0078997-Liu1">[50]</xref> found that the <italic>SPCH/MUTE/FAMA</italic> genes from <italic>Arabidopsis thaliana</italic>, <italic>Oryza sativa</italic> and <italic>Zea mays</italic> formed three monophyletic groups, respectively, although the functions of SPCH and MUTE from <italic>Oryza</italic> are somewhat divergent from their homologs in <italic>Arabidopsis</italic>. In contrast, by sampling <italic>Arabidopsis</italic>, <italic>Populus</italic>, <italic>Oryza</italic>, and <italic>Physcomitrella</italic>, Peterson <italic>et al</italic>. <xref ref-type="bibr" rid="pone.0078997-Peterson1">[33]</xref> found that the angiosperm <italic>SPCH</italic> and <italic>MUTE</italic> genes formed a clade sister to a clade comprising the angiosperm <italic>FAMA</italic> genes and the <italic>Physcomitrella</italic> bHLH Ia members. In addition, MacAlister and Bergmann <xref ref-type="bibr" rid="pone.0078997-MacAlister2">[31]</xref> constructed a phylogeny of the SMF genes based on a sampling of nine angiosperms plus <italic>Selaginella moellendorffii</italic> and <italic>Physcomitrella patens</italic> with complete genome sequences and a EST sequence (<italic>Pgl</italic>‘<italic>SPCH’</italic> [<italic>PgSPCH</italic>]) from the conifer <italic>Picea glauca</italic>. They found that the angiosperm <italic>SPCH</italic>, <italic>MUTE</italic> and <italic>FAMA</italic> genes formed monophyletic groups, respectively, and <italic>SPCH</italic> was closer to <italic>FAMA</italic> than <italic>MUTE</italic>. Particularly, <italic>SmoSMF2</italic> (<italic>SmSMF2</italic>) and <italic>SmoSMF3</italic> (<italic>SmSMF3</italic>) (Ia members of <italic>Selaginella</italic>) were sister to the angiosperm <italic>FAMA</italic> genes, while <italic>SmoSMF1</italic> (<italic>SmSMF1</italic>) (also an Ia member of <italic>Selaginella</italic>), <italic>PpaSMF1</italic> (<italic>PpSMF2</italic>) and <italic>PpaSMF2</italic> (<italic>PpSMF2</italic>) (Ia members of <italic>Physcomitrella</italic>) were sister to the angiosperm <italic>MUTE</italic> genes with strong bootstrap supports, and <italic>PglSPCH</italic> was sister to the angiosperm <italic>MUTE</italic> genes with a weak support (49%). This phylogeny also seems to be supported by the domain architectures of the genes that they designated <xref ref-type="bibr" rid="pone.0078997-MacAlister2">[31]</xref>. Therefore, as discussed above, the evolutionary history and patterns of the bHLH Ia genes involved in stomatal development are still greatly controversial, especially the phylogenetic positions of the Ia genes from basal land plants and gymnosperms.</p><p>In the present study, putative homologs of the bHLH Ia genes were identified from 49 species representing four major lineages of land plants (bryophyte, lycophyte, gymnosperm, and angiosperm). Because previous phylogenetic studies indicate that the bHLH Ia genes are monophyletic <xref ref-type="bibr" rid="pone.0078997-MacAlister1">[26]</xref>, <xref ref-type="bibr" rid="pone.0078997-Pires1">[29]</xref>, <xref ref-type="bibr" rid="pone.0078997-ToledoOrtiz1">[34]</xref>, <xref ref-type="bibr" rid="pone.0078997-Li1">[35]</xref> and non-bHLH amino acid motifs are highly conserved in each bHLH subgroup <xref ref-type="bibr" rid="pone.0078997-Pires1">[29]</xref>, <xref ref-type="bibr" rid="pone.0078997-Heim1">[30]</xref>, we only include the bHLH Ia members in this study in order to avoid incorrect phylogenetic topology that could be caused by improper alignment between different subgroups. Also, considering that the Ia genes first appeared in <italic>Physcomitrella patens</italic>, the two Ia members from this species (<italic>PpaSMF1/2</italic>) were used as functional outgroups in the phylogenetic analysis. The ML trees based respectively on the amino acid sequences and the first plus second codon positions both support that the Ia genes from vascular plants could be divided into three clades. That is, Clade A comprises the SMF genes from <italic>Selaginella moellendorffii</italic> and the <italic>FAMA</italic> homologs from seed plants (gymnosperm and angiosperm); Clade B includes two monophyletic sister groups, i.e., the gymnosperm “<italic>SPCH</italic>” genes and the angiosperm <italic>MUTE</italic> and <italic>SPCH</italic> genes; and Clade C includes other Ia members from gymnosperms and angiosperms that could not be grouped with <italic>SPCH/MUTE/FAMA</italic> (<xref ref-type="fig" rid="pone-0078997-g001">Fig. 1A</xref>). The unrooted ML tree also supports the three clades and a close relationship between the Ia genes from <italic>Physcomitrella patens</italic> and Clade A (<xref ref-type="fig" rid="pone-0078997-g001">Fig. 1B</xref>).</p><p>The <italic>SPCH</italic>, <italic>MUTE</italic> and <italic>FAMA</italic> genes of angiosperms form monophyletic groups, respectively, which is corroborated by their different gene structures (<xref ref-type="fig" rid="pone-0078997-g001">Figs. 1</xref>, <xref ref-type="fig" rid="pone-0078997-g002">2</xref>). All <italic>SPCH</italic> genes have the unique and conserved MPKTD domain, although with different lengths, and this domain is important for regulating the SPCH activity in response to phosphorylation by the MAP kinases <xref ref-type="bibr" rid="pone.0078997-Lampard1">[51]</xref>. In addition, except <italic>PvuSPCH1</italic> and <italic>LusSPCH3</italic>, all other SPCHs have a conserved position of stop codon. The <italic>MUTE</italic> genes have a unique conserved region (V) <xref ref-type="bibr" rid="pone.0078997-MacAlister2">[31]</xref>, and lack some residues preceding the bHLH domain that are present in all the other bHLH Ia members with various lengths. In contrast, the <italic>FAMA</italic> genes have high AA sequence similarity (<xref ref-type="table" rid="pone-0078997-t004">Table 4</xref>), and harbor three unique domains (I, II and IV) (<xref ref-type="fig" rid="pone-0078997-g002">Fig. 2</xref>).</p><p>For gymnosperms, the <italic>FAMA</italic> and ‘<italic>SPCH’</italic> genes were found in the EST databases of <italic>Ginkgo biloba</italic>, <italic>Picea glauca</italic>, <italic>Pinus banksiana</italic> and <italic>Pinus taeda</italic> (<xref ref-type="table" rid="pone-0078997-t002">Tables 2</xref>, <xref ref-type="supplementary-material" rid="pone.0078997.s003">S1</xref>), and were successfully PCR-amplified from <italic>Picea breweriana</italic>, <italic>P. jezoensis</italic> and <italic>P. sitchensis</italic>. We also tried to amplify the ‘<italic>MUTE’</italic> genes from gymnosperms using different primers, but failed. Moreover, we blasted the draft genome sequence of <italic>Picea abies</italic> just released, which could have covered 96% of the protein-coding genes of this species <xref ref-type="bibr" rid="pone.0078997-Nystedt1">[52]</xref>, and found four bHLH Ia genes [ConGenIE:MA_57244g0010, ConGenIE:MA_686524g0010, ConGenIE:MA_120602g0010 and ConGenIE:MA_130776g0010] (<ext-link ext-link-type="uri" xlink:href="http://congenie.org">http://congenie.org</ext-link>). When the four genes were added into our dataset, the generated tree is topologically identical to <xref ref-type="fig" rid="pone-0078997-g001">Figure 1</xref> and indicates that MA_57244g0010 is a <italic>FAMA</italic> homolog and MA_120602g0010 is a ‘<italic>SPCH’</italic> homolog. The other two genes belong to other Ias (Tree not shown). Therefore, all available evidence strongly suggests that the <italic>MUTE</italic> gene does not occur in gymnosperms.</p><p>Undoubtedly, as suggested by MacAlister and Bergmann <xref ref-type="bibr" rid="pone.0078997-MacAlister2">[31]</xref>, the gymnosperm <italic>FAMA</italic> genes are sister to the angiosperm <italic>FAMA</italic> genes (<xref ref-type="fig" rid="pone-0078997-g001">Fig. 1</xref>), and the sister relationship is also supported by the high similarities of gene structures (<xref ref-type="fig" rid="pone-0078997-g002">Fig. 2</xref>). However, the gymnosperm ‘<italic>SPCH’</italic> genes are sister to a clade comprising the angiosperm <italic>SPCH</italic> and <italic>MUTE</italic> genes with 100% bootstrap support, although they are very similar to the angiosperm <italic>SPCH</italic> genes in structure, especially the presence of the N-terminal sequence before the bHLH domain and the partial MPKTD domain (<xref ref-type="fig" rid="pone-0078997-g002">Fig. 2</xref>). By comparing the AA sequences of the bHLH and SMF domains, we found that the gymnosperm ‘<italic>SPCH’</italic> genes are similar to the angiosperm <italic>SPCH</italic> sequences in some sites, but are identical to the angiosperm <italic>MUTE</italic> sequences in some other sites (<xref ref-type="fig" rid="pone-0078997-g003">Fig. 3</xref>). Additionally, deletion of the MPKTD domain of SPCH can generate proteins with functions similar to MUTE <xref ref-type="bibr" rid="pone.0078997-OhashiIto1">[27]</xref>, <xref ref-type="bibr" rid="pone.0078997-Lampard1">[51]</xref>. Therefore, we deduce that the gymnosperm ‘<italic>SPCH’</italic> genes may be closely related to and function as the common ancestor of the angiosperm <italic>SPCH/MUTE</italic> genes, and the <italic>MUTE</italic> genes could be derived from the ancestral copy by losing the MPKTD domain and the N-terminal sequences before the bHLH domain. This inference is also consistent with the reconstruction of ancestral gene structure (<xref ref-type="supplementary-material" rid="pone.0078997.s001">Fig. S1</xref>).</p><p>All bHLH Ia genes from <italic>S. moellendorffii</italic> are located in Clade A (FAMA), which is in line with Pires and Dolan <xref ref-type="bibr" rid="pone.0078997-Pires1">[29]</xref> and Peterson <italic>et al</italic>. <xref ref-type="bibr" rid="pone.0078997-Peterson1">[33]</xref> but different from MacAlister and Bergmann <xref ref-type="bibr" rid="pone.0078997-MacAlister2">[31]</xref>. In MacAlister and Bergmann <xref ref-type="bibr" rid="pone.0078997-MacAlister2">[31]</xref>, SmoSMF1 was placed sister to the angiosperm <italic>MUTE</italic> genes whereas SmoSMF2–3 were sister to the angiosperm <italic>FAMA</italic> genes. Comparing sequences of the conserved domains and structures of these genes, we found that all members from <italic>Selaginella</italic> have the pre-SMF domain (IV) and the unique region prior to the bHLH domain (II) (<xref ref-type="fig" rid="pone-0078997-g002">Fig. 2</xref>). Meanwhile, SmoSMF1 and SmoSMF2 have the conserved region in the N-terminus (I) that occurs in most angiosperm <italic>FAMA</italic> genes. This information, together with the high AA sequence similarity among <italic>SmoSMF</italic>s and the <italic>FAMA</italic> genes of gymnosperms and angiosperms (data not shown) and the phylogenetic reconstruction (<xref ref-type="fig" rid="pone-0078997-g001">Fig. 1</xref>), strongly suggests that all bHLH Ia members of <italic>Selaginella</italic> have close relationships with the <italic>FAMA</italic> genes. In addition, the function of the <italic>FAMA</italic> genes might be ancestral in bHLH Ia subgroup because <italic>PpaSMFs</italic> and <italic>SmoSMFs</italic> from the basal land plants are structurally identical and closely related to the <italic>FAMA</italic> genes of gymnosperms and angiosperms (<xref ref-type="fig" rid="pone-0078997-g001">Figs. 1</xref>, <xref ref-type="fig" rid="pone-0078997-g002">2</xref>, <xref ref-type="supplementary-material" rid="pone.0078997.s001">S1</xref>).</p><p>Among the bHLH Ia genes from angiosperms, the <italic>FAMA</italic> genes are more conserved than the other ones, showing not only the lowest nucleotide diversity (<italic>Pi</italic> = 0.21573), but also the lowest nonsynonymous substitution rate (<italic>Ka</italic> = 0.05844) and <italic>Ka/Ks</italic> ratio (<italic>ω</italic> = 0.091). The <italic>SPCH</italic> and <italic>MUTE</italic> genes have similar values of nucleotide diversity, nonsynonymous and synonymous substitution rates, and <italic>Ka/Ks</italic> (<xref ref-type="table" rid="pone-0078997-t004">Table 4</xref>). Therefore, the <italic>SPCH/MUTE/FAMA</italic> genes might have experienced different selective pressures, although the fact that no site or branch was detected to be under positive or relaxed selection using Fitmodel and codeml suggests conserved functions of all of them (<xref ref-type="table" rid="pone-0078997-t003">Table 3</xref>, <xref ref-type="supplementary-material" rid="pone.0078997.s005">Table S3</xref>). Nevertheless, the functional shift could have occurred repeatedly in the <italic>SPCH</italic> genes, considering that more copies of them are maintained in some angiosperm species (<xref ref-type="table" rid="pone-0078997-t001">Table 1</xref>), and that the functional divergence of different <italic>SPCH</italic> copies has been reported in <italic>Oryza sativa</italic> and <italic>Zea mays</italic><xref ref-type="bibr" rid="pone.0078997-Liu1">[50]</xref>. It cannot be ruled out that the multiple conspecific copies of the bHLH Ia subgroup, especially for the <italic>SPCH/MUTE/FAMA</italic> genes, were caused by ancient or recent genome duplications (<ext-link ext-link-type="uri" xlink:href="http://chibba.agtec.uga.edu/duplication/">http://chibba.agtec.uga.edu/duplication/</ext-link>). For example, some species that have experienced recent whole-genome duplication (WGD), such as <italic>Brassica rapa</italic>, <italic>Glycine max</italic>, <italic>Musa acuminate</italic>, <italic>Populus trichocarpa</italic>, <italic>Solanum lycopersicum</italic>, <italic>Solanum tuberosum</italic> and <italic>Zea mays</italic>, harbor more bHLH Ia genes. However, recent WGD cannot explain why more <italic>SPCH</italic> genes are retained than the <italic>MUTE</italic> and <italic>FAMA</italic> genes in some species (<xref ref-type="table" rid="pone-0078997-t001">Table 1</xref>). Until now, no functional studies have been performed for other bHLH Ia genes (<xref ref-type="fig" rid="pone-0078997-g001">Fig. 1A</xref>, Clade C). These genes experienced frequent duplication and extinction events, both ancient and recent, and thus it would be interesting to investigate their functional differentiation in the future (<xref ref-type="supplementary-material" rid="pone.0078997.s002">Fig. S2</xref>).</p></sec><sec id="s4b"><title>Evolution of Stomatal Development in Land Plants</title><p>The developmental process of stomata has evolved and become complex with the evolution of land plants, especially the occurrence of amplifying divisions <xref ref-type="bibr" rid="pone.0078997-McAdam2">[14]</xref>. This process has been well studied in <italic>Arabidopsis</italic>, which includes three main steps, i.e., asymmetric entry divisions of the meristemoid mother cells (MMCs) to create meristemoids (M), self-renew via amplifying divisions or direct differentiation of meristemoids into GMCs, and symmetrical divisions of GMCs to form GCs <xref ref-type="bibr" rid="pone.0078997-Pillitteri2">[53]</xref>. However, in mosses and lycophytes, stomata develop by a simple process, in which a single asymmetric or symmetric division is followed by differentiation of a GMC, then GCs ( <xref ref-type="bibr" rid="pone.0078997-Payne1">[54]</xref>,reviewed by <xref ref-type="bibr" rid="pone.0078997-Rudall1">[55]</xref>). For ferns, an epidermal cell may go through one or two asymmetric divisions, then differentiates into a GMC, and finally into GCs <xref ref-type="bibr" rid="pone.0078997-Apostolakos1">[56]</xref>. In gymnosperms, different stomatal development patterns have been reported. The meristemoid divides once symmetrically to generate a GMC that then develops into GCs in <italic>Pinus</italic><xref ref-type="bibr" rid="pone.0078997-Johnson1">[57]</xref>, but it is interesting that <italic>Ginkgo biloba</italic>, a living fossil of gymnosperms, possesses both asymmetric divisions in perigenous neighbor cells like grasses and amplifying divisions within the stomatal lineage like <italic>Arabidopsis</italic><xref ref-type="bibr" rid="pone.0078997-Rudall2">[58]</xref>. The basal angiosperms and dicots are basically similar to <italic>A. thaliana</italic> in stomatal development, although many of them show some differences in amplifying divisions <xref ref-type="bibr" rid="pone.0078997-Carpenter1">[59]</xref>. In monocots, the formation of two guard cells needs two steps if not considering the subsidiary cells, i.e., one asymmetric division to generate a GMC, and a symmetric division to form two guard cells <xref ref-type="bibr" rid="pone.0078997-Sack2">[60]</xref>, <xref ref-type="bibr" rid="pone.0078997-Stebbins1">[61]</xref>. The presence or absence of at least one asymmetric division in the cell lineage leading to the guard mother cell is a potential key factor in land-plant evolution. However, it is unclear which kind of division (symmetric or asymmetric) is ancestral in land plants <xref ref-type="bibr" rid="pone.0078997-Rudall1">[55]</xref>. More works are needed on the stomatal development of early land plants.</p><p>Based on the above information, it is obvious that the basic developmental process of stomata has not greatly changed in land plants if we do not consider the formation of the neighbor cells. At least two steps are necessary and conserved. One is the formation of GMC by one or two symmetric or asymmetric divisions, and the other is the formation of two GCs by a symmetric division. The two steps are directly mediated by the <italic>MUTE</italic> and <italic>FAMA</italic> genes, which has been proved by the experiments that PpaSMF1 and PpaSMF2 can partially rescue the <italic>fama</italic> and <italic>mute</italic> phenotypes <xref ref-type="bibr" rid="pone.0078997-MacAlister2">[31]</xref>, and by the fact that the <italic>MUTE</italic> and <italic>FAMA</italic> genes are functionally conserved in the monocots <italic>Oryza sativa</italic> and <italic>Zea mays</italic><xref ref-type="bibr" rid="pone.0078997-Liu1">[50]</xref>, and the eudicot <italic>A. thaliana</italic> (Reviewed by <xref ref-type="bibr" rid="pone.0078997-Pillitteri2">[53]</xref>).</p><p>The <italic>SPCH</italic> gene controls the first asymmetric division of protodermal cells to initiate the stomatal lineage in <italic>Arabidopsis</italic>. However, functions of the two <italic>SPCH</italic> homologs from <italic>Oryza sativa</italic> (<italic>OsaSPCH1</italic>, <italic>OsaSPCH2</italic>) have somewhat diverged. The <italic>OsaSPCH2</italic> plays a role in promoting the early events of the stomatal development whereas <italic>OsaSPCH1</italic> does not <xref ref-type="bibr" rid="pone.0078997-Liu1">[50]</xref>. In addition, the <italic>SPCH</italic> genes of angiosperms have experienced more duplication events and harbor more copies than the <italic>MUTE</italic> and <italic>FAMA</italic> genes (<xref ref-type="table" rid="pone-0078997-t001">Table 1</xref>). All this information, together with the variation of the stomatal development in the entry division <xref ref-type="bibr" rid="pone.0078997-Peterson1">[33]</xref>, <xref ref-type="bibr" rid="pone.0078997-Rudall2">[58]</xref>, implies that the <italic>SPCH</italic> genes could have contributed greatly to the diversity of stomatal development in angiosperms.</p><p>MacAlister and Bergmann <xref ref-type="bibr" rid="pone.0078997-MacAlister2">[31]</xref> constructed a model to explain the developmental complexity of the stomatal lineage. In their model, a single multifunctional bHLH Ia gene was originally responsible for both specification of GMC identity and GC differentiation in early land plants. Then, the ancestral Ia gene was duplicated into two genes, <italic>MUTE</italic> for GMC identity and <italic>FAMA</italic> for GC differentiation. Finally, a third Ia member, <italic>SPCH</italic>, originated from another gene duplication, which allows for the typical stomatal lineage in angiosperms, including amplifying divisions. This model was based on the phylogeny of the <italic>SPCH/MUTE/FAMA</italic> genes constructed in their study that sampled nine angiosperms, <italic>Picea glauca</italic>, <italic>Physcomitrella patens</italic> and <italic>Selaginella moellendorffii</italic>, and particularly based on their finding that PpaSMF1, PpaSMF2 and SmoSMF1 grouped with the angiosperm MUTEs. However, in that phylogeny, it is difficult to understand that <italic>PglSPCH</italic>, one Ia member from the gymnosperm <italic>P. glauca</italic>, was grouped with the angiosperm <italic>SPCH</italic> genes, although with only 49% bootstrap support, while no <italic>MUTE</italic> gene was found in gymnosperms. Furthermore, that phylogeny is topologically different from the phylogenies constructed in the present and all other previous studies <xref ref-type="bibr" rid="pone.0078997-Pires1">[29]</xref>, <xref ref-type="bibr" rid="pone.0078997-Peterson1">[33]</xref>, <xref ref-type="bibr" rid="pone.0078997-Li1">[35]</xref>, <xref ref-type="bibr" rid="pone.0078997-Liu1">[50]</xref>. Therefore, it seems incredible that <italic>MUTE</italic> originated before <italic>SPCH</italic> if the <italic>MUTE</italic> gene does not occur in gymnosperms.</p><p>In the present study, all bHLH Ia genes from the two basal land plants <italic>Physcomitrella</italic> and <italic>Selaginella</italic> show close relationships with the <italic>FAMA</italic> genes of gymnosperms and angiosperms, and the angiosperm <italic>SPCH</italic> and <italic>MUTE</italic> genes form a clade sister to the gymnosperm ‘<italic>SPCH</italic>’ genes (<xref ref-type="fig" rid="pone-0078997-g001">Fig. 1</xref>). Hence, we agree with MacAlister and Bergmann <xref ref-type="bibr" rid="pone.0078997-MacAlister2">[31]</xref> that an ancestral Ia gene with multifunction could control the specification of GMC identity and GC differentiation in basal land plants. However, we argue that <italic>SPCH</italic> originated earlier than <italic>MUTE</italic> based on the gene phylogeny and structure. The <italic>SPCH</italic> and <italic>MUTE</italic> genes are easily to be identified because they have a MPKTD domain and a truncated N-terminus, respectively. Theoretically, it is easier for <italic>MUTE</italic> to lose the N-terminus than for <italic>SPCH</italic> to gain the MPKTD domain <xref ref-type="bibr" rid="pone.0078997-MacAlister2">[31]</xref>. However, the MPKTD domain could have originated before the divergence of gymnosperms and angiosperms, given the fact that the gymnosperm ‘<italic>SPCH</italic>’ genes have a truncated MPKTD domain whereas the <italic>MUTE</italic> genes are only found in angiosperms. Furthermore, the partial deletion of the MPKTD in <italic>SPCH</italic> generates a protein that more resembles <italic>MUTE</italic> in function <xref ref-type="bibr" rid="pone.0078997-Lampard1">[51]</xref>, also supporting that the <italic>MUTE</italic> gene might have derived from the ancestor of the <italic>SPCH</italic> gene by loss of the MPKTD domain. To reveal the evolutionary history of the MPKTD domain, more species of lycophytes and ferns need to be studied.</p><p>Based on the phylogeny and structure of the bHLH Ia genes (<xref ref-type="fig" rid="pone-0078997-g001">Figs. 1</xref>, <xref ref-type="fig" rid="pone-0078997-g002">2</xref>), we deduce that a single multifunctional Ia gene responsible for the stomatal development originated in early land plants. Then, the ancestral Ia gene gave rise to two genes by a duplication in the ancestor of seed plants, one keeping the function of <italic>FAMA</italic> while the other (like the gymnosperm ‘<italic>SPCH</italic>’ genes) acquiring the MPKTD domain and having a dual function of <italic>SPCH</italic> and <italic>MUTE</italic>. Finally, the ‘<italic>SPCH</italic>’ gene evolved into the two genes <italic>SPCH</italic> and <italic>MUTE</italic> by another duplication in the ancestor of angiosperms. The <italic>SPCH</italic> retained the MPKTD domain, whereas the <italic>MUTE</italic> lost it, and got a later start codon at the beginning of the bHLH domain due to the disruption of the initial start codon (<xref ref-type="fig" rid="pone-0078997-g004">Fig. 4</xref>). One may argue that the gymnosperm <italic>FAMA</italic> genes could have multifunction as the <italic>FAMA</italic> genes in basal land plants. However, it is clear that the gymnosperm ‘<italic>SPCH</italic>’ genes are sister to the angiosperm <italic>SPCH</italic>-<italic>MUTE</italic> genes (<xref ref-type="fig" rid="pone-0078997-g001">Fig. 1</xref>). For a better understanding of functions of the <italic>SPCH/MUTE/FAMA</italic> genes, more functional studies should be done in non-flowering vascular plants in the future.</p><fig id="pone-0078997-g004" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0078997.g004</object-id><label>Figure 4</label><caption><title>A predictive model of the stomatal development with the evolution of land plants.</title><p>(A) A single, multifunctional Ia gene responsible for the formation of both guard mother cell (GMC) and guard cells (GCs). (B) An ancestral ‘<italic>SPCH</italic>’ that originally occurred in gymnosperms by a gene duplication has a dual function of <italic>MUTE</italic> and <italic>SPCH</italic> and allows for the divergence between <italic>FAMA</italic> and ‘<italic>SPCH’</italic>. (C) The ‘<italic>SPCH</italic>’ gene evolved into the two genes <italic>SPCH</italic> and <italic>MUTE</italic> by another duplication in the ancestor of angiosperms.</p></caption><graphic xlink:href="pone.0078997.g004"></graphic></fig></sec></sec><sec id="s5"><title>Conclusions</title><p>In this study, we revealed the evolutionary relationships of three bHLH Ia genes (<italic>SPCH</italic>, <italic>MUTE</italic> and <italic>FAMA</italic>) of land plants that mediate sequential steps of stomatal development based on a wide sampling. The most interesting findings include the close relationship between the Ia genes from basal land plants (<italic>Physcomitrella</italic> and <italic>Selaginella</italic>) and the <italic>FAMA</italic> genes of seed plants, and the sister relationship between the gymnosperm ‘<italic>SPCH</italic>’ genes and the angiosperm <italic>SPCH</italic> and <italic>MUTE</italic> genes. Also, we proposed a model for the correlation between the evolution of stomatal development and the genes involved in this developmental process in land plants. However, only a few of bHLH Ia genes of gymnosperms that are available were included in this study, and almost nothing is known about the functions of these genes. It would be of great interest to investigate evolution and function of the ‘<italic>SPCH</italic>’ and <italic>FAMA</italic> genes in more gymnosperms in the future.</p></sec><sec sec-type="supplementary-material" id="s6"><title>Supporting Information</title><supplementary-material content-type="local-data" id="pone.0078997.s001"><label>Figure S1</label><caption><p><bold>Phylogeny of the bHLH Ia genes with a reconstruction of ancestral gene structure.</bold></p><p>(PDF)</p></caption><media xlink:href="pone.0078997.s001.pdf"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pone.0078997.s002"><label>Figure S2</label><caption><p><bold>Maximum-likelihood (ML) trees of the bHLH Ia genes.</bold> A, A rooted ML tree based on amino acid sequences with the bHLH Ia genes from <italic>Physcomitrella patens</italic> as outgroups. B, A rooted tree based on nucleotide sequences with the bHLH Ia genes from <italic>P. patens</italic> as outgroups. C, An unrooted ML tree of the Ia genes constructed based on amino acid sequences. Numbers above branches refer to bootstrap values higher than 50%. The stars denote some inferred duplication events. Gene names are shown in <xref ref-type="supplementary-material" rid="pone.0078997.s003">Table S1</xref>. Ang, angiosperms; Gym, gymnosperms; Smo, <italic>moellendorffii</italic>; Ppa, <italic>Physcomitrella patens</italic>.</p><p>(PDF)</p></caption><media xlink:href="pone.0078997.s002.pdf"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pone.0078997.s003"><label>Table S1</label><caption><p><bold>The sources of the Ia members of the bHLH transcriptional factors we analyzed.</bold></p><p>(XLS)</p></caption><media xlink:href="pone.0078997.s003.xls"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pone.0078997.s004"><label>Table S2</label><caption><p><bold>The primers used in this study.</bold> * represent the primers used in the gene amplification.</p><p>(DOC)</p></caption><media xlink:href="pone.0078997.s004.doc"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pone.0078997.s005"><label>Table S3</label><caption><p><bold>Results of the site and branch-site analyses of the </bold><bold><italic>SPCH/MUTE/SPCH</italic></bold><bold> genes.</bold></p><p>(XLS)</p></caption><media xlink:href="pone.0078997.s005.xls"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec></body><back><ack><p>The authors thank Prof. Brandon S. Gaut for his careful reading and insightful comments that greatly improved the manuscript, Dr. Dong-Mei Guo for her assistance in the Fitmodel analysis, Ming-Ming Wang for his assistance in retrieving the bHLH Ia genes in gymnosperms and Ms. Wan-Wing Jin in the DNA sequencing.</p></ack><ref-list><title>References</title><ref id="pone.0078997-Bateman1"><label>1</label><mixed-citation publication-type="journal"><name><surname>Bateman</surname><given-names>RM</given-names></name>, <name><surname>Crane</surname><given-names>PR</given-names></name>, <name><surname>DiMichele</surname><given-names>WA</given-names></name>, <name><surname>Kenrick</surname><given-names>PR</given-names></name>, <name><surname>Rowe</surname><given-names>NP</given-names></name>, <etal>et al</etal> (<year>1998</year>) <article-title>Early evolution of land plants: Phylogeny, physiology, and ecology of the primary terrestrial radiation</article-title>. <source>Annu Rev Ecol Syst</source><volume>29</volume>: <fpage>263</fpage>–<lpage>292</lpage></mixed-citation></ref><ref id="pone.0078997-Delaux1"><label>2</label><mixed-citation publication-type="journal"><name><surname>Delaux</surname><given-names>PM</given-names></name>, <name><surname>Nanda</surname><given-names>AK</given-names></name>, <name><surname>Mathe</surname><given-names>C</given-names></name>, <name><surname>Sejalon-Delmas</surname><given-names>N</given-names></name>, <name><surname>Dunand</surname><given-names>C</given-names></name> (<year>2012</year>) <article-title>Molecular and biochemical aspects of plant terrestrialization</article-title>. <source>Perspect Plant Ecol Evol Syst</source><volume>14</volume>: <fpage>49</fpage>–<lpage>59</lpage></mixed-citation></ref><ref id="pone.0078997-Edwards1"><label>3</label><mixed-citation publication-type="journal"><name><surname>Edwards</surname><given-names>D</given-names></name>, <name><surname>Kerp</surname><given-names>H</given-names></name>, <name><surname>Hass</surname><given-names>H</given-names></name> (<year>1998</year>) <article-title>Stomata in early land plants: an anatomical and ecophysiological approach</article-title>. <source>J Exp Bot</source><volume>49</volume>: <fpage>255</fpage>–<lpage>278</lpage></mixed-citation></ref><ref id="pone.0078997-Raven1"><label>4</label><mixed-citation publication-type="journal"><name><surname>Raven</surname><given-names>JA</given-names></name> (<year>2002</year>) <article-title>Selection pressures on stomatal evolution</article-title>. <source>New Phytol</source><volume>153</volume>: <fpage>371</fpage>–<lpage>386</lpage></mixed-citation></ref><ref id="pone.0078997-Sack1"><label>5</label><mixed-citation publication-type="journal"><name><surname>Sack</surname><given-names>FD</given-names></name>, <name><surname>Paolillo</surname><given-names>DJ</given-names></name> (<year>1985</year>) <article-title>Incomplete cytokinesis in <italic>Funaria</italic> stomata</article-title>. <source>Am J Bot</source><volume>72</volume>: <fpage>1325</fpage>–<lpage>1333</lpage></mixed-citation></ref><ref id="pone.0078997-Torii1"><label>6</label><mixed-citation publication-type="journal"><name><surname>Torii</surname><given-names>KU</given-names></name> (<year>2007</year>) <article-title>Stomatal patterning and guard cell differentiation</article-title>. <source>Plant Cell Monogr</source><volume>9</volume>: <fpage>343</fpage>–<lpage>359</lpage></mixed-citation></ref><ref id="pone.0078997-Beerling1"><label>7</label><mixed-citation publication-type="journal"><name><surname>Beerling</surname><given-names>DJ</given-names></name>, <name><surname>Franks</surname><given-names>PJ</given-names></name> (<year>2009</year>) <article-title>Evolution of stomatal function in 'lower' land plants</article-title>. <source>New Phytol</source><volume>183</volume>: <fpage>921</fpage>–<lpage>925</lpage><pub-id pub-id-type="pmid">19702775</pub-id></mixed-citation></ref><ref id="pone.0078997-Chater1"><label>8</label><mixed-citation publication-type="journal"><name><surname>Chater</surname><given-names>C</given-names></name>, <name><surname>Kamisugi</surname><given-names>Y</given-names></name>, <name><surname>Movahedi</surname><given-names>M</given-names></name>, <name><surname>Fleming</surname><given-names>A</given-names></name>, <name><surname>Cuming</surname><given-names>AC</given-names></name>, <etal>et al</etal> (<year>2011</year>) <article-title>Regulatory mechanism controlling stomatal behavior conserved across 400 million years of land plant evolution</article-title>. <source>Curr Biol</source><volume>21</volume>: <fpage>1025</fpage>–<lpage>1029</lpage><pub-id pub-id-type="pmid">21658944</pub-id></mixed-citation></ref><ref id="pone.0078997-Haworth1"><label>9</label><mixed-citation publication-type="journal"><name><surname>Haworth</surname><given-names>M</given-names></name>, <name><surname>Elliott-Kingston</surname><given-names>C</given-names></name>, <name><surname>McElwain</surname><given-names>JC</given-names></name> (<year>2011</year>) <article-title>Stomatal control as a driver of plant evolution</article-title>. <source>J Exp Bot</source><volume>62</volume>: <fpage>2419</fpage>–<lpage>2423</lpage><pub-id pub-id-type="pmid">21576397</pub-id></mixed-citation></ref><ref id="pone.0078997-McAdam1"><label>10</label><mixed-citation publication-type="journal"><name><surname>McAdam</surname><given-names>SAM</given-names></name>, <name><surname>Brodribb</surname><given-names>TJ</given-names></name>, <name><surname>Ross</surname><given-names>JJ</given-names></name>, <name><surname>Jordan</surname><given-names>GJ</given-names></name> (<year>2011</year>) <article-title>Augmentation of abscisic acid (ABA) levels by drought does not induce short-term stomatal sensitivity to CO<sub>2</sub> in two divergent conifer species</article-title>. <source>J Exp Bot</source><volume>62</volume>: <fpage>195</fpage>–<lpage>203</lpage><pub-id pub-id-type="pmid">20797996</pub-id></mixed-citation></ref><ref id="pone.0078997-Doi1"><label>11</label><mixed-citation publication-type="journal"><name><surname>Doi</surname><given-names>M</given-names></name>, <name><surname>Shimazaki</surname><given-names>K</given-names></name> (<year>2008</year>) <article-title>The stomata of the fern <italic>Adiantum capillus-veneris</italic> do Not respond to CO<sub>2</sub> in the dark and open by photosynthesis in guard cells</article-title>. <source>Plant Physiol</source><volume>147</volume>: <fpage>922</fpage>–<lpage>930</lpage><pub-id pub-id-type="pmid">18467462</pub-id></mixed-citation></ref><ref id="pone.0078997-Brodribb1"><label>12</label><mixed-citation publication-type="journal"><name><surname>Brodribb</surname><given-names>TJ</given-names></name>, <name><surname>McAdam</surname><given-names>SAM</given-names></name>, <name><surname>Jordan</surname><given-names>GJ</given-names></name>, <name><surname>Feild</surname><given-names>TS</given-names></name> (<year>2009</year>) <article-title>Evolution of stomatal responsiveness to CO<sub>2</sub> and optimization of water-use efficiency among land plants</article-title>. <source>New Phytol</source><volume>183</volume>: <fpage>839</fpage>–<lpage>847</lpage><pub-id pub-id-type="pmid">19402882</pub-id></mixed-citation></ref><ref id="pone.0078997-Brodribb2"><label>13</label><mixed-citation publication-type="journal"><name><surname>Brodribb</surname><given-names>TJ</given-names></name>, <name><surname>McAdam</surname><given-names>SAM</given-names></name> (<year>2011</year>) <article-title>Passive origins of stomatal control in vascular plants</article-title>. <source>Science</source><volume>331</volume>: <fpage>582</fpage>–<lpage>585</lpage><pub-id pub-id-type="pmid">21163966</pub-id></mixed-citation></ref><ref id="pone.0078997-McAdam2"><label>14</label><mixed-citation publication-type="journal"><name><surname>McAdam</surname><given-names>SA</given-names></name>, <name><surname>Brodribb</surname><given-names>TJ</given-names></name> (<year>2012</year>) <article-title>Stomatal innovation and the rise of seed plants</article-title>. <source>Ecol Lett</source><volume>15</volume>: <fpage>1</fpage>–<lpage>8</lpage><pub-id pub-id-type="pmid">22017636</pub-id></mixed-citation></ref><ref id="pone.0078997-deBoer1"><label>15</label><mixed-citation publication-type="journal"><name><surname>de Boer</surname><given-names>HJ</given-names></name>, <name><surname>Eppinga</surname><given-names>MB</given-names></name>, <name><surname>Wassen</surname><given-names>MJ</given-names></name>, <name><surname>Dekker</surname><given-names>SC</given-names></name> (<year>2012</year>) <article-title>A critical transition in leaf evolution facilitated the Cretaceous angiosperm revolution</article-title>. <source>Nat Commun</source><volume>3</volume>: <fpage>1221</fpage><pub-id pub-id-type="pmid">23187621</pub-id></mixed-citation></ref><ref id="pone.0078997-Robinson1"><label>16</label><mixed-citation publication-type="journal"><name><surname>Robinson</surname><given-names>JM</given-names></name> (<year>1994</year>) <article-title>Speculations on carbon-dioxide starvation, Late Tertiary evolution of stomatal regulation and floristic modernization</article-title>. <source>Plant Cell Environ</source><volume>17</volume>: <fpage>345</fpage>–<lpage>354</lpage></mixed-citation></ref><ref id="pone.0078997-Franks1"><label>17</label><mixed-citation publication-type="journal"><name><surname>Franks</surname><given-names>PJ</given-names></name>, <name><surname>Beerling</surname><given-names>DJ</given-names></name> (<year>2009</year>) <article-title>CO<sub>2</sub>-forced evolution of plant gas exchange capacity and water-use efficiency over the Phanerozoic</article-title>. <source>Geobiology</source><volume>7</volume>: <fpage>227</fpage>–<lpage>236</lpage><pub-id pub-id-type="pmid">19338614</pub-id></mixed-citation></ref><ref id="pone.0078997-Doi2"><label>18</label><mixed-citation publication-type="journal"><name><surname>Doi</surname><given-names>M</given-names></name>, <name><surname>Wada</surname><given-names>M</given-names></name>, <name><surname>Shimazaki</surname><given-names>K</given-names></name> (<year>2006</year>) <article-title>The fern <italic>Adiantum capillus-veneris</italic> lacks stomatal responses to blue light</article-title>. <source>Plant Cell Physiol</source><volume>47</volume>: <fpage>748</fpage>–<lpage>755</lpage><pub-id pub-id-type="pmid">16621842</pub-id></mixed-citation></ref><ref id="pone.0078997-Croxdale1"><label>19</label><mixed-citation publication-type="journal"><name><surname>Croxdale</surname><given-names>JL</given-names></name> (<year>2000</year>) <article-title>Stomatal patterning in angiosperms</article-title>. <source>Am J Bot</source><volume>87</volume>: <fpage>1069</fpage>–<lpage>1080</lpage><pub-id pub-id-type="pmid">10947991</pub-id></mixed-citation></ref><ref id="pone.0078997-Darwin1"><label>20</label><mixed-citation publication-type="journal"><name><surname>Darwin</surname><given-names>F</given-names></name> (<year>1898</year>) <article-title>Observations on stomata</article-title>. <source>Phil Trans R Soc Lond B</source><volume>190</volume>: <fpage>531</fpage>–<lpage>621</lpage></mixed-citation></ref><ref id="pone.0078997-Berry1"><label>21</label><mixed-citation publication-type="journal"><name><surname>Berry</surname><given-names>JA</given-names></name>, <name><surname>Beerling</surname><given-names>DJ</given-names></name>, <name><surname>Franks</surname><given-names>PJ</given-names></name> (<year>2010</year>) <article-title>Stomata: key players in the earth system, past and present</article-title>. <source>Curr Opin Plant Biol</source><volume>13</volume>: <fpage>232</fpage>–<lpage>239</lpage></mixed-citation></ref><ref id="pone.0078997-Ruszala1"><label>22</label><mixed-citation publication-type="journal"><name><surname>Ruszala</surname><given-names>EM</given-names></name>, <name><surname>Beerling</surname><given-names>DJ</given-names></name>, <name><surname>Franks</surname><given-names>PJ</given-names></name>, <name><surname>Chater</surname><given-names>C</given-names></name>, <name><surname>Casson</surname><given-names>SA</given-names></name>, <etal>et al</etal> (<year>2011</year>) <article-title>Land plants acquired active stomatal control early in their evolutionary history</article-title>. <source>Curr Biol</source><volume>21</volume>: <fpage>1030</fpage>–<lpage>1035</lpage><pub-id pub-id-type="pmid">21658945</pub-id></mixed-citation></ref><ref id="pone.0078997-McAdam3"><label>23</label><mixed-citation publication-type="journal"><name><surname>McAdam</surname><given-names>SA</given-names></name>, <name><surname>Brodribb</surname><given-names>TJ</given-names></name> (<year>2013</year>) <article-title>Ancestral stomatal control results in a canalization of fern and lycophyte adaptation to drought</article-title>. <source>New Phytol</source><volume>198</volume>: <fpage>429</fpage>–<lpage>441</lpage><pub-id pub-id-type="pmid">23421706</pub-id></mixed-citation></ref><ref id="pone.0078997-Kanaoka1"><label>24</label><mixed-citation publication-type="journal"><name><surname>Kanaoka</surname><given-names>MM</given-names></name>, <name><surname>Pillitteri</surname><given-names>LJ</given-names></name>, <name><surname>Fujii</surname><given-names>H</given-names></name>, <name><surname>Yoshida</surname><given-names>Y</given-names></name>, <name><surname>Bogenschutz</surname><given-names>NL</given-names></name>, <etal>et al</etal> (<year>2008</year>) <article-title><italic>SCREAM/ICE1</italic> and <italic>SCREAM2</italic> specify three cell-state transitional steps leading to <italic>Arabidopsis</italic> stomatal differentiation</article-title>. <source>Plant Cell</source><volume>20</volume>: <fpage>1775</fpage>–<lpage>1185</lpage><pub-id pub-id-type="pmid">18641265</pub-id></mixed-citation></ref><ref id="pone.0078997-Pillitteri1"><label>25</label><mixed-citation publication-type="journal"><name><surname>Pillitteri</surname><given-names>LJ</given-names></name>, <name><surname>Torii</surname><given-names>KU</given-names></name> (<year>2007</year>) <article-title>Breaking the silence: three bHLH proteins direct cell-fate decisions during stomatal development</article-title>. <source>BioEssays</source><volume>29</volume>: <fpage>861</fpage>–<lpage>870</lpage><pub-id pub-id-type="pmid">17691100</pub-id></mixed-citation></ref><ref id="pone.0078997-MacAlister1"><label>26</label><mixed-citation publication-type="journal"><name><surname>MacAlister</surname><given-names>CA</given-names></name>, <name><surname>Ohashi-Ito</surname><given-names>K</given-names></name>, <name><surname>Bergmann</surname><given-names>DC</given-names></name> (<year>2007</year>) <article-title>Transcription factor control of asymmetric cell divisions that establish the stomatal lineage</article-title>. <source>Nature</source><volume>445</volume>: <fpage>537</fpage>–<lpage>540</lpage><pub-id pub-id-type="pmid">17183265</pub-id></mixed-citation></ref><ref id="pone.0078997-OhashiIto1"><label>27</label><mixed-citation publication-type="journal"><name><surname>Ohashi-Ito</surname><given-names>K</given-names></name>, <name><surname>Bergmann</surname><given-names>DC</given-names></name> (<year>2006</year>) <article-title><italic>Arabidopsis</italic> FAMA controls the final proliferation/differentiation switch during stomatal development</article-title>. <source>Plant Cell</source><volume>18</volume>: <fpage>2493</fpage>–<lpage>2505</lpage><pub-id pub-id-type="pmid">17088607</pub-id></mixed-citation></ref><ref id="pone.0078997-Gudesblat1"><label>28</label><mixed-citation publication-type="journal"><name><surname>Gudesblat</surname><given-names>GE</given-names></name>, <name><surname>Schneider-Pizon</surname><given-names>J</given-names></name>, <name><surname>Betti</surname><given-names>C</given-names></name>, <name><surname>Mayerhofer</surname><given-names>J</given-names></name>, <name><surname>Vanhoutte</surname><given-names>I</given-names></name>, <etal>et al</etal> (<year>2012</year>) <article-title>SPEECHLESS integrates brassinosteroid and stomata signalling pathways</article-title>. <source>Nat Cell Biol</source><volume>14</volume>: <fpage>548</fpage>–<lpage>554</lpage><pub-id pub-id-type="pmid">22466366</pub-id></mixed-citation></ref><ref id="pone.0078997-Pires1"><label>29</label><mixed-citation publication-type="journal"><name><surname>Pires</surname><given-names>N</given-names></name>, <name><surname>Dolan</surname><given-names>L</given-names></name> (<year>2010</year>) <article-title>Origin and diversification of basic-helix-loop-helix proteins in plants</article-title>. <source>Mol Biol Evol</source><volume>27</volume>: <fpage>862</fpage>–<lpage>874</lpage><pub-id pub-id-type="pmid">19942615</pub-id></mixed-citation></ref><ref id="pone.0078997-Heim1"><label>30</label><mixed-citation publication-type="journal"><name><surname>Heim</surname><given-names>MA</given-names></name>, <name><surname>Jakoby</surname><given-names>M</given-names></name>, <name><surname>Werber</surname><given-names>M</given-names></name>, <name><surname>Martin</surname><given-names>C</given-names></name>, <name><surname>Weisshaar</surname><given-names>B</given-names></name>, <etal>et al</etal> (<year>2003</year>) <article-title>The basic helix-loop-helix transcription factor family in plants: a genome-wide study of protein structure and functional diversity</article-title>. <source>Mol Biol Evol</source><volume>20</volume>: <fpage>735</fpage>–<lpage>747</lpage><pub-id pub-id-type="pmid">12679534</pub-id></mixed-citation></ref><ref id="pone.0078997-MacAlister2"><label>31</label><mixed-citation publication-type="journal"><name><surname>MacAlister</surname><given-names>CA</given-names></name>, <name><surname>Bergmann</surname><given-names>DC</given-names></name> (<year>2011</year>) <article-title>Sequence and function of basic helix-loop-helix proteins required for stomatal development in Arabidopsis are deeply conserved in land plants</article-title>. <source>Evol Dev</source><volume>13</volume>: <fpage>182</fpage>–<lpage>192</lpage><pub-id pub-id-type="pmid">21410874</pub-id></mixed-citation></ref><ref id="pone.0078997-Vaten1"><label>32</label><mixed-citation publication-type="journal"><name><surname>Vaten</surname><given-names>A</given-names></name>, <name><surname>Bergmann</surname><given-names>DC</given-names></name> (<year>2012</year>) <article-title>Mechanisms of stomatal development: an evolutionary view</article-title>. <source>EvoDevo</source><volume>3</volume>: <fpage>11</fpage><pub-id pub-id-type="pmid">22691547</pub-id></mixed-citation></ref><ref id="pone.0078997-Peterson1"><label>33</label><mixed-citation publication-type="journal"><name><surname>Peterson</surname><given-names>KM</given-names></name>, <name><surname>Rychel</surname><given-names>AL</given-names></name>, <name><surname>Torii</surname><given-names>KU</given-names></name> (<year>2010</year>) <article-title>Out of the mouths of plants: the molecular basis of the evolution and diversity of stomatal development</article-title>. <source>Plant Cell</source><volume>22</volume>: <fpage>296</fpage>–<lpage>306</lpage><pub-id pub-id-type="pmid">20179138</pub-id></mixed-citation></ref><ref id="pone.0078997-ToledoOrtiz1"><label>34</label><mixed-citation publication-type="journal"><name><surname>Toledo-Ortiz</surname><given-names>G</given-names></name>, <name><surname>Huq</surname><given-names>E</given-names></name>, <name><surname>Quail</surname><given-names>PH</given-names></name> (<year>2003</year>) <article-title>The <italic>Arabidopsis</italic> basic/helix-loop-helix transcription factor family</article-title>. <source>Plant Cell</source><volume>15</volume>: <fpage>1749</fpage>–<lpage>1770</lpage><pub-id pub-id-type="pmid">12897250</pub-id></mixed-citation></ref><ref id="pone.0078997-Li1"><label>35</label><mixed-citation publication-type="journal"><name><surname>Li</surname><given-names>X</given-names></name>, <name><surname>Duan</surname><given-names>X</given-names></name>, <name><surname>Jiang</surname><given-names>H</given-names></name>, <name><surname>Sun</surname><given-names>Y</given-names></name>, <name><surname>Tang</surname><given-names>Y</given-names></name>, <etal>et al</etal> (<year>2006</year>) <article-title>Genome-wide analysis of basic/helix-loop-helix transcription factor family in rice and Arabidopsis</article-title>. <source>Plant Physiol</source><volume>141</volume>: <fpage>1167</fpage>–<lpage>1184</lpage><pub-id pub-id-type="pmid">16896230</pub-id></mixed-citation></ref><ref id="pone.0078997-Doyle1"><label>36</label><mixed-citation publication-type="journal"><name><surname>Doyle</surname><given-names>JA</given-names></name>, <name><surname>Doyle</surname><given-names>JL</given-names></name> (<year>1987</year>) <article-title>A rapid DNA isolation procedure for small quantities of fresh leaf tissue</article-title>. <source>Phytochem Bull</source><volume>19</volume>: <fpage>11</fpage>–<lpage>15</lpage></mixed-citation></ref><ref id="pone.0078997-Rogers1"><label>37</label><mixed-citation publication-type="other">Rogers SO, Bendich AJ (1988) Extraction of DNA from plant tissues. In: Gelvin SB, Schilperoort RA, editors. Plant Molecular Biology Manual. Boston, MA: Kluwer Academic Publishers. 1–10.</mixed-citation></ref><ref id="pone.0078997-Guo1"><label>38</label><mixed-citation publication-type="journal"><name><surname>Guo</surname><given-names>D–M</given-names></name>, <name><surname>Ran</surname><given-names>J–H</given-names></name>, <name><surname>Wang</surname><given-names>X-Q</given-names></name> (<year>2010</year>) <article-title>Evolution of the cinnamyl/sinapyl alcohol dehydrogenase (CAD/SAD) gene family: The emergence of real lignin is associated with the origin of <italic>bona fide</italic> CAD</article-title>. <source>J Mol Evol</source><volume>71</volume>: <fpage>202</fpage>–<lpage>218</lpage><pub-id pub-id-type="pmid">20721545</pub-id></mixed-citation></ref><ref id="pone.0078997-Ran1"><label>39</label><mixed-citation publication-type="journal"><name><surname>Ran</surname><given-names>J–H</given-names></name>, <name><surname>Gao</surname><given-names>H</given-names></name>, <name><surname>Wang</surname><given-names>X-Q</given-names></name> (<year>2010</year>) <article-title>Fast evolution of the retroprocessed mitochondrial <italic>rps3</italic> gene in Conifer II and further evidence for the phylogeny of gymnosperms</article-title>. <source>Mol Phylogenet Evol</source><volume>54</volume>: <fpage>136</fpage>–<lpage>149</lpage><pub-id pub-id-type="pmid">19761858</pub-id></mixed-citation></ref><ref id="pone.0078997-Thompson1"><label>40</label><mixed-citation publication-type="journal"><name><surname>Thompson</surname><given-names>JD</given-names></name>, <name><surname>Gibson</surname><given-names>TJ</given-names></name>, <name><surname>Plewniak</surname><given-names>F</given-names></name>, <name><surname>Jeanmougin</surname><given-names>F</given-names></name>, <name><surname>Higgins</surname><given-names>DG</given-names></name> (<year>1997</year>) <article-title>The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools</article-title>. <source>Nucleic Acids Res</source><volume>25</volume>: <fpage>4876</fpage>–<lpage>4882</lpage><pub-id pub-id-type="pmid">9396791</pub-id></mixed-citation></ref><ref id="pone.0078997-Xia1"><label>41</label><mixed-citation publication-type="journal"><name><surname>Xia</surname><given-names>X</given-names></name>, <name><surname>Xie</surname><given-names>Z</given-names></name> (<year>2001</year>) <article-title>DAMBE: software package for data analysis in molecular biology and evolution</article-title>. <source>J Hered</source><volume>92</volume>: <fpage>371</fpage>–<lpage>373</lpage><pub-id pub-id-type="pmid">11535656</pub-id></mixed-citation></ref><ref id="pone.0078997-Darriba1"><label>42</label><mixed-citation publication-type="journal"><name><surname>Darriba</surname><given-names>D</given-names></name>, <name><surname>Taboada</surname><given-names>GL</given-names></name>, <name><surname>Doallo</surname><given-names>R</given-names></name>, <name><surname>Posada</surname><given-names>D</given-names></name> (<year>2012</year>) <article-title>jModelTest 2: more models, new heuristics and parallel computing</article-title>. <source>Nat Methods</source><volume>9</volume>: <fpage>772</fpage><pub-id pub-id-type="pmid">22847109</pub-id></mixed-citation></ref><ref id="pone.0078997-Darriba2"><label>43</label><mixed-citation publication-type="journal"><name><surname>Darriba</surname><given-names>D</given-names></name>, <name><surname>Taboada</surname><given-names>GL</given-names></name>, <name><surname>Doallo</surname><given-names>R</given-names></name>, <name><surname>Posada</surname><given-names>D</given-names></name> (<year>2011</year>) <article-title>ProtTest 3: fast selection of best-fit models of protein evolution</article-title>. <source>Bioinformatics</source><volume>27</volume>: <fpage>1164</fpage>–<lpage>1165</lpage><pub-id pub-id-type="pmid">21335321</pub-id></mixed-citation></ref><ref id="pone.0078997-Huelsenbeck1"><label>44</label><mixed-citation publication-type="journal"><name><surname>Huelsenbeck</surname><given-names>JP</given-names></name>, <name><surname>Ronquist</surname><given-names>F</given-names></name> (<year>2001</year>) <article-title>MRBAYES: Bayesian inference of phylogenetic trees</article-title>. <source>Bioinformatics</source><volume>17</volume>: <fpage>754</fpage>–<lpage>755</lpage><pub-id pub-id-type="pmid">11524383</pub-id></mixed-citation></ref><ref id="pone.0078997-Guindon1"><label>45</label><mixed-citation publication-type="journal"><name><surname>Guindon</surname><given-names>S</given-names></name>, <name><surname>Gascuel</surname><given-names>O</given-names></name> (<year>2003</year>) <article-title>A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood</article-title>. <source>Syst Biol</source><volume>52</volume>: <fpage>696</fpage>–<lpage>704</lpage><pub-id pub-id-type="pmid">14530136</pub-id></mixed-citation></ref><ref id="pone.0078997-Guindon2"><label>46</label><mixed-citation publication-type="journal"><name><surname>Guindon</surname><given-names>S</given-names></name>, <name><surname>Rodrigo</surname><given-names>AG</given-names></name>, <name><surname>Dyer</surname><given-names>KA</given-names></name>, <name><surname>Huelsenbeck</surname><given-names>JP</given-names></name> (<year>2004</year>) <article-title>Modeling the site-specific variation of selection patterns along lineages</article-title>. <source>Proc Natl Acad Sci U S A</source><volume>101</volume>: <fpage>12957</fpage>–<lpage>12962</lpage><pub-id pub-id-type="pmid">15326304</pub-id></mixed-citation></ref><ref id="pone.0078997-Yang1"><label>47</label><mixed-citation publication-type="journal"><name><surname>Yang</surname><given-names>Z</given-names></name> (<year>2007</year>) <article-title>PAML 4: phylogenetic analysis by maximum likelihood</article-title>. <source>Mol Biol Evol</source><volume>24</volume>: <fpage>1586</fpage>–<lpage>1591</lpage><pub-id pub-id-type="pmid">17483113</pub-id></mixed-citation></ref><ref id="pone.0078997-Librado1"><label>48</label><mixed-citation publication-type="journal"><name><surname>Librado</surname><given-names>P</given-names></name>, <name><surname>Rozas</surname><given-names>J</given-names></name> (<year>2009</year>) <article-title>DnaSP v5: a software for comprehensive analysis of DNA polymorphism data</article-title>. <source>Bioinformatics</source><volume>25</volume>: <fpage>1451</fpage>–<lpage>1452</lpage><pub-id pub-id-type="pmid">19346325</pub-id></mixed-citation></ref><ref id="pone.0078997-Maddison1"><label>49</label><mixed-citation publication-type="other">Maddison WP, Maddison DR Mesquite: a modular system for evolutionary analysis. Version 2.75 [<ext-link ext-link-type="uri" xlink:href="http://mesquiteproject.org">http://mesquiteproject.org</ext-link>].</mixed-citation></ref><ref id="pone.0078997-Liu1"><label>50</label><mixed-citation publication-type="journal"><name><surname>Liu</surname><given-names>T</given-names></name>, <name><surname>Ohashi-Ito</surname><given-names>K</given-names></name>, <name><surname>Bergmann</surname><given-names>DC</given-names></name> (<year>2009</year>) <article-title>Orthologs of <italic>Arabidopsis thaliana</italic> stomatal bHLH genes and regulation of stomatal development in grasses</article-title>. <source>Development</source><volume>136</volume>: <fpage>2265</fpage>–<lpage>2276</lpage><pub-id pub-id-type="pmid">19502487</pub-id></mixed-citation></ref><ref id="pone.0078997-Lampard1"><label>51</label><mixed-citation publication-type="journal"><name><surname>Lampard</surname><given-names>GR</given-names></name>, <name><surname>MacAlister</surname><given-names>CA</given-names></name>, <name><surname>Bergmann</surname><given-names>DC</given-names></name> (<year>2008</year>) <article-title><italic>Arabidopsis</italic> stomatal initiation is controlled by MAPK-mediated regulation of the bHLH SPEECHLESS</article-title>. <source>Science</source><volume>322</volume>: <fpage>1113</fpage>–<lpage>1116</lpage><pub-id pub-id-type="pmid">19008449</pub-id></mixed-citation></ref><ref id="pone.0078997-Nystedt1"><label>52</label><mixed-citation publication-type="journal"><name><surname>Nystedt</surname><given-names>B</given-names></name>, <name><surname>Street</surname><given-names>NR</given-names></name>, <name><surname>Wetterbom</surname><given-names>A</given-names></name>, <name><surname>Zuccolo</surname><given-names>A</given-names></name>, <name><surname>Lin</surname><given-names>YC</given-names></name>, <etal>et al</etal> (<year>2013</year>) <article-title>The Norway spruce genome sequence and conifer genome evolution</article-title>. <source>Nature</source><volume>497</volume>: <fpage>579</fpage>–<lpage>584</lpage><pub-id pub-id-type="pmid">23698360</pub-id></mixed-citation></ref><ref id="pone.0078997-Pillitteri2"><label>53</label><mixed-citation publication-type="journal"><name><surname>Pillitteri</surname><given-names>LJ</given-names></name>, <name><surname>Torii</surname><given-names>KU</given-names></name> (<year>2012</year>) <article-title>Mechanisms of stomatal development</article-title>. <source>Annu Rev Plant Biol</source><volume>63</volume>: <fpage>591</fpage>–<lpage>614</lpage><pub-id pub-id-type="pmid">22404473</pub-id></mixed-citation></ref><ref id="pone.0078997-Payne1"><label>54</label><mixed-citation publication-type="journal"><name><surname>Payne</surname><given-names>WW</given-names></name> (<year>1979</year>) <article-title>Stomatal patterns in embryophytes - their evolution, ontogeny and interpretation</article-title>. <source>Taxon</source><volume>28</volume>: <fpage>117</fpage>–<lpage>132</lpage></mixed-citation></ref><ref id="pone.0078997-Rudall1"><label>55</label><mixed-citation publication-type="other">Rudall PJ, Hilton J, Bateman RM (2013) Several developmental and morphogenetic factors govern the evolution of stomatal patterning in land plants. New Phytol DOI: 10.1111/nph.12406.</mixed-citation></ref><ref id="pone.0078997-Apostolakos1"><label>56</label><mixed-citation publication-type="journal"><name><surname>Apostolakos</surname><given-names>P</given-names></name>, <name><surname>Panteris</surname><given-names>E</given-names></name>, <name><surname>Galatis</surname><given-names>B</given-names></name> (<year>1997</year>) <article-title>Microtubule and actin filament organization during stomatal morphogenesis in the fern <italic>Asplenium nidus</italic>. 1. Guard cell mother cell</article-title>. <source>Protoplasma</source><volume>198</volume>: <fpage>93</fpage>–<lpage>106</lpage></mixed-citation></ref><ref id="pone.0078997-Johnson1"><label>57</label><mixed-citation publication-type="journal"><name><surname>Johnson</surname><given-names>RW</given-names></name>, <name><surname>Riding</surname><given-names>RT</given-names></name> (<year>1981</year>) <article-title>Structure and ontogeny of the stomatal complex in <italic>Pinus strobus</italic> L. and <italic>Pinus banksiana</italic> Lamb</article-title>. <source>Am J Bot</source><volume>68</volume>: <fpage>260</fpage>–<lpage>268</lpage></mixed-citation></ref><ref id="pone.0078997-Rudall2"><label>58</label><mixed-citation publication-type="journal"><name><surname>Rudall</surname><given-names>PJ</given-names></name>, <name><surname>Rowland</surname><given-names>A</given-names></name>, <name><surname>Bateman</surname><given-names>RM</given-names></name> (<year>2012</year>) <article-title>Ultrastructure of stomatal development in <italic>Ginkgo biloba</italic></article-title>. <source>Int J Plant Sci</source><volume>173</volume>: <fpage>849</fpage>–<lpage>860</lpage></mixed-citation></ref><ref id="pone.0078997-Carpenter1"><label>59</label><mixed-citation publication-type="journal"><name><surname>Carpenter</surname><given-names>KJ</given-names></name> (<year>2005</year>) <article-title>Stomatal architecture and evolution in basal angiosperms</article-title>. <source>Am J Bot</source><volume>92</volume>: <fpage>1595</fpage>–<lpage>1615</lpage><pub-id pub-id-type="pmid">21646077</pub-id></mixed-citation></ref><ref id="pone.0078997-Sack2"><label>60</label><mixed-citation publication-type="journal"><name><surname>Sack</surname><given-names>FD</given-names></name> (<year>1994</year>) <article-title>Structure of the stomatal complex of the monocot <italic>Flagellaria indica</italic></article-title>. <source>Am J Bot</source><volume>81</volume>: <fpage>339</fpage>–<lpage>344</lpage></mixed-citation></ref><ref id="pone.0078997-Stebbins1"><label>61</label><mixed-citation publication-type="journal"><name><surname>Stebbins</surname><given-names>GL</given-names></name>, <name><surname>Jain</surname><given-names>SK</given-names></name> (<year>1960</year>) <article-title>Developmental studies of cell differentiation in the epidermis of monocotyledons: I. Allium, rhoeo, and commelina</article-title>. <source>Dev Biol</source><volume>2</volume>: <fpage>409</fpage>–<lpage>426</lpage></mixed-citation></ref></ref-list></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Wicri/Bois/explor/OrangerV1/Data/Pmc/Corpus
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 001175 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd -nk 001175 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Wicri/Bois
|area= OrangerV1
|flux= Pmc
|étape= Corpus
|type= RBID
|clé= PMC:3823973
|texte= Evolution of the bHLH Genes Involved in Stomatal Development: Implications for the Expansion of Developmental Complexity of Stomata in Land Plants
}}
Pour générer des pages wiki
HfdIndexSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/RBID.i -Sk "pubmed:24244399" \
| HfdSelect -Kh $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd \
| NlmPubMed2Wicri -a OrangerV1
| This area was generated with Dilib version V0.6.25. |  |