Botanical Studies (2010) 51: 119-126. SYstematics Rubovietnamia nonggangensis (Rubiaceae), a new species from China Feng-JuanMOU1,2andDian-XiangZHANG1,* 1South China Botanical Garden, The Chinese Academy of Sciences, Guangzhou 510650, P.R. China 2Southwest Forestry University, Kunming 650224, P.R. China (ReceivedDecember8,2008;AcceptedJune18,2009) ABSTRACT. AnewRubiaceaespecies,Rubovietnamia nonggangensisF.J.Mou&D.X.Zhang,isde- scribedandillustratedfromGuangxi,China.Thesomaticchromosomenumber(2n =22)andpollenmorphol- ogyofthespeciesarereported.Thenewspeciesischaracterizedprominentlybyhavingdensehairsonmany organs,suchasyoungbranches,leaves,andinflorescences,enlargedandfoliaceouscalyxlobescaducous after anthesis, and indehiscent and globose fruits with persistent yellowish annular floral disks on the apex. AllmorphologicaldatasupportthatitbelongstothegenusRubovietnamia,agenusdistributedinVietnamand southern China, currently classified in the tribe Gardenieae of the subfamily Ixoroideae. Keywords:Chromosomenumber;Gardenieae;Molecularphylogeny;Newspecies;Pollenmorphology; Rubiaceae;Rubovietnamia; Rubovietnamia nonggangensis. INTRODUCTION aspeciescharacterizedwithcreamyandfragrantcorollas was found. Based on the leaf-like calyx lobes and ThegenusRubovietnamia Tirveng. was first described indumentum,thespecimenswereputativelyreferredtoas fromnorthernVietnambasedonasinglespeciesRubovi- anewspeciesofthegenusRubovietnamia. etnamia aristataTirveng.(Tirvengadum,1998).Recently, ithasbeenrecordedfromGuangxiandYunnaninChina MATERIALS AND METHODS (Zhangetal.,2007).Rubovietnamiaiscloselyrelatedto VidalasiaTirveng.inthetribeGardenieae(Rubiaceae), Morphology andischaracterizedbytheindehiscent,few-tomany- Themorphologicalcharactersofthenewspecieswere seededandrelativelylargefruits,containingajuicypulp studiedbasedonlivingspecimensintheirnaturalhabitats formedfromtheseptumand/ortheplacenta(Robbrecht andonplantsgrowinginSCBG(SouthChinaBotanical andPuff,1986).Tirvengadum(1998)describedthesole Garden,theChineseAcademyofSciences)andonspeci- speciesofthegenusasfollows:ashrubwithreddish mensfromherbariainChina(IBK[GuangxiInstituteof brownbark,itsleafapexoftenmarkedlyaristatedueto Botany]andIBSC[theSouthChinaBotanicalGarden, prolongationandthemainnerveprojectedbeyondtheacu- CAS]).Therelevantliteratureconcerningthegenuswas menontheundersurfaceofthelimb;thedeltoidstipules alsoreviewed.Thefieldstudieswerecarriedoutthree connateatbase,caudate-acuminateatapex,subcoriaceous, timesfromMaytoJuly2008inGuangxiProvince,China. providedwithhairsandfewsmallcolletersmostlytowards the base inside; the inflorescence a 2-4-flowered cyme or Palynology withasolitaryflower;bractsandbracteolesminuteand stipule-like;calyxfoliaceouswithalargebaseandaristate Pollensamplescollectedfromdriedspecimen(F. J. atapex;corollatubemuchlongerthanthelobes,cylin- Mou 209-1,IBSC)werepreparedbyacetolysis10min dricalwitharingofshorthairstowardsthebaseinside; at95-100°C(Erdtman,1960).Theunacetolyzedand anthersattachedtothemiddleofthecorollatubeandhalf- acetolyzedpollenswererinsedtwicerespectivelywithul- exserted;styleslenderandcoveredwithshorthairswith trasoundin70%ethanolandstoredin70%ethanol.After bifid and grooved stigma; ovary 1-celled; fruit globose and dryinginair,pollengrainsweresputter-coatedwithgold- smooth;seedsovoidtosubglobose. palladiumandexaminedusingaJSE-6360LVSEMata voltageof10KV.Morethantwentypollengrainswere Duringafieldtripin2008intheNonggangNature measuredforacetolyzedpollenofthespecies.Theter- Reserve,LongzhouCounty,GuangxiProvinceinChina, minologyfollowsmainlyErdtman(1952)andFaegriand Iversen(1989). *Correspondingauthor:E-mail:[email protected]. 120 Botanical Studies, Vol. 51, 2010 Cytology Table 1.SpeciesandGeneBankaccessionnumbers(including Mitoticchromosomeswereinvestigatedbyusingroot previouslypublishedsequences). tipsfromplantscultivatedinthegreenhouseofSCBG. Taxa trnL-F rps16 Activelygrowingroottipswerepre-treatedwith0.002 Aidia micrantha (K.Schum.)F.White AF2009741 AF2010281 mol/L8-hydroxyquinolinesolutionwhileshieldedfrom 1 light for about 1.5 h at 4°C, washed with distilled water, Aoranthe castaneofulva (S.Moore)Somers AF200977 fixedinfreshcoldCarnoy’sFluid(ethanol:aceticacid Atractocarpus stipularis (F.Muell.)Puttock AF2009791 AF2010311 solution=3:1)inamixtureofwaterandicefor2-3hat Benkara malabarica (Lam.)Tirveng. AF2009811 AF2010331 4°C, thrice washed with distilled water, and later hydro- Calochone redingii (DeWild.)Keay AF2009861 AF2010361 lyzedin1mol/Lhydrochloricacidat60°Cfor7-8min. Afterthreemorerinsesindistilledwater,roottipswere Casasia calophylla A.Rich. AF2009871 AF2010371 stainedinCarbolFuchsinsolutionformorethan15min Catunaregam spinosa (Thunb.)Tirveng. AF2009881 AF2010381 andthensquashedontheslideforlightmicroscopy. Ceriscoides sessiliflora (Wall.exKurz) AF2009891 AF2010391 Tirveng. Molecular phylogeny Deccania pubescens (Roth)Tirveng. AF2009911 AF2010411 TotalDNAwasisolatedfromfreshleavesofRubovi- 1 1 etnamia nonggangensiscollectedfromplantsgrowingin Duperrea pavettifolia (Kurz)Pitard AF200993 AF201042 thegreenhouseinSCBG(F. J. Mou 230,IBSC)usingthe Euclinia longifloraSalis. AF2009951 AF2010431 modified 2 × CTAB method after leaf tissue was ground Gardenia volkensii subsp.spathulifolia AF2009961 AF2010441 inliquidnitrogen(DoyleandDoyle,1987).Thisstudy Stapf.&Hutch. usedtwosequencesofcpDNA,the trnL-Fregion(Taberlet 1 1 etal.,1991)andthe rps16geneintron(Oxelmanetal., Genipa americanaL. AF200997 AF201045 1997).ThetrnL-F region was amplified with primers c (5´ Kailarsenia ochreata (F.Muell.)Puttock AF2010021 AF2010491 -CGAAATCGGTAGACGCTACG-3´) and f (5´-ATTT- Macrosphyra longistyla (DC.)Hook.f.ex AF2010041 AF2010511 GAACTGGTGACACGAG-3´) (Taberlet et al., 1991). The Hiern rps16 intron was amplified as described by Oxelman et al. 1 1 (1997) with primers rpsF (5´-GTGGTAGAAAGCAAC- Massularia acuminate(G.Don)Bullockex AF201005 AF201052 Hoyle GTGCGACTT- 3´) and rpsR2 (5´-TCGGGATCGAA- CATCAATTGCAAC- 3´). The PCR amplification used Morelia senegalensis A.Rich.exDC. AF2010071 AF2010551 a 5 min denaturing step at 94°C followed by 35 cycles of Oligocodon cunliffeae (Wernham)Keay AF2010081 AF2010561 denaturing for 1 min at 94°C, primer annealing for 1.5 Oxyceros longiflorus (Lam.)Yamazaki AF2010101 AF2010581 minat52°C,andelongationfor1.5minat72°C,witha 1 1 final 10 min elongation step at 72°C. The PCR products Porterandia crosbyi (Burkill)A.C.Sm.&S. AF201012 AF201059 weresequencedusingthesameprimercombinationasfor Darwin PCRamplifications.Sequencespublishedinaprevious Preussiodora sulphurea (K.Schum.)Keay AF2010131 AF2010601 studyofRubiaceae(AnderssonandRova,1999;Persson, 1 1 Rosenbergiodendron densiflorum (K. AF201014 AF201061 2000;Rovaetal.,2002;BremerandEriksson,2009)were Schum.)Fagerl. downloadedfromGenBankandusedinthepresentstudy 2 2 (Table1). Rothmannia capensis Thunb. AM117340 AM117384 InconsiderationofthephylogenyofGardenieaebased Rubovietnamia nonggangensis F.J.Mou& GQ251031 GQ249894 onchloroplastDNAsequencesfromthe rpsl6intronand D.X.Zhang trnL(U.AA)-F(GAA)intergenicspacersequence(Anders- Sherbourniasp. AF2010171 AF2010641 sonandRova,1999;Persson,2000;Rova,etal.,2002; Sphinctanthus microphyllus K.Schum. AF2010201 AF2010661 BremerandEriksson,2009),thirtytaxa(includingthenew 1 1 species)includedin“theCoreGardenieaeclade”were Sukunia longipes A.C.Sm. AF201022 AF201068 selectedasingroupsandthreetaxa(Polysphaeriasp., Tamilnadia uliginosa (Retz.)Tirveng. AF2010231 AF2010691 Psydrax odorataandIxora killipii)fromthetribesOc- Tarennoidea wallichii (Hook.f)Tirveng.& AF2010251 AF2010701 totropideae,Vanguerieae,andIxoreae,respectively,were Sastre chosenasoutgroups.Aparsimonyanalysiswasperformed Trukia fitzalanii (F.Muell.)Fosberg AF2010271 AF2010721 usingaheuristicsearchwith100randomsequenceaddi- tionreplicates,andtreeswerelimitedto10,000during Ixora killipii Standl. AF2010011 AF1526593 eachreplicate.Gapsweretreatedasmissingdata.Boot- Polysphaeriasp. AF2010111 AF1526553 strapvalues(BP)oftheinternalnodeswereobtainedwith Psydrax odorata(Forst.f)A.C.Smith&S. AF0040314 AF1526563 500replicates.Ineachreplicate,weperformed10random P.Darwin sequenceadditionreplicatesfollowedbyatreebisection- 1 2 3 reconnection(TBR)swappingalgorithmandkeepingno Note: Persson(2000); BremeandEriksson(2009); Rovaet 4 morethan1000treesperreplicate. al.(2002); AnderssonandRova(1999). MOU and ZHANG — Rubovietnamia nonggangensis, a new species from China 121 RESULTS AND DISCUSSION trudings(Figure2A-E).Thepollengrainsofthenewspe- ciesarethusdistinguishedfromRubovietnamiaaristata, Morphology whichhasheterogeneouslyreticulatesexineornamentation ThecollectionsfromGuangxiProvincebearsasuper- (Tirvengadum,1998).Theexineornamentationofpollen ficial resemblance to Rubovietnamia aristata Tirveng.in isusuallyreticulate,butalsofoveolate,rugulate,perforate havingcymoseinflorescences,awell-developedcorolla andpsilateinthesubtribe Gardeniinae(Persson,1993). tubeandlobesandglobosefruits(Figure1A-G;Table2). However,theycanbedistinguishedeasilyfromR. aristata Cytology inhavingmuchbroaderfoliaceouscalyxlobes,andthe Themitoticchromosomenumberofthenewspecies leaves,youngbranchesandflowers(includingpedicles, is2n=22(Figure3F),whichiscongruentwiththatof ovary,stylesandcalyx)denselycoveredwithhairs.These mosttaxareportedinthetribeGardenieae,includingthe specimensarethusproposedasanewspecieshere. generaCatunaregam, Massularia,Rosenbergiodendron, Rothmannia(Gadella,1982;Kiehn,1985;KiehnandLor- Palynology ence,1996), Sukunia(Kiehn,1996.)and Gardenia(Bhat- Thepollengrainsofthenewspeciesaremonads,ob- tacharyya,1958),while2n=20or22havebeenreported