TThhee LLeepipdiopdteorpoltoegircaollSoocgieitycal Society ooff JJaapapnan uctts,kTrans.Iepid.Soc.Jlipan60 (4):293-297,March 2010 Genetjcdistances Oeneis O.polixenesO,.bore O. among norna, and metissa (Satyri dbaasee)d on mitochondrial DNA (NDI) partial sequences Takao IToH*, Kazuhiko HiRANo and Kazuo WATANABE GraduateSchool IntegrateAdrts ScienceH,iroshimaUniyersity, of and Higashi-Hiroshima City,739-8521 Japan Abstract Partia ]sequences (4S2 bp) of mitochendrial DNA (mtDNA) IVDJ of Oeneis norna and Oeneis ntetissa have been determined and compared with reperted NDI sequences of Oeneis polix- enes, O. bore (O. taygete) and O. uhteri. I tis inferr efdrom the comparison of parti amltDNA se- quences that O. hore, O. polixenes, O. norna and O. melissa are distinc tspecies to one another and their ancestors were separated at almost the same age, It is mentioned also that at presen tthere is no clear evidence showing that O. non!a is inhabjting North America including Alaska, whieh cen- tradicts the description sgiven in a number of illustrat erdeference books on Japanese butterflies, Key words Oeneis noma, Oeneis polixenes, Oeneis bore, Oeneis metissa, Mitochondrial DNA (NDI) genes, geneti dcistance. Introduction The species of genus Oeneis Ellr efound in the north of the New and Old Worlds. All the species are dull colored, medium in size and principa] lfyound in Arctic or alpjne habitat ass well as in taiga fores t.In Japan two Oeneis species, O. noma and O. melissa, distribute, both of which are regarded as high-altitu dspeecies inhabitin glimite dareas above the tim- ber]in e.The fbrrne rspecies inhabit sthe Hjda meuntain chain (th eNorthern Japan Alps) and Mt, Yatsugatak ein Honshu Island ,while the latte irnhabit sthe Daisets umountains and the Hidaka mountain chain in Hoklcaid oIsland .In most of the illustrat reedference books published after 1975 in Japan, it is describe dthat O. norna ranges both Eurasia and North America (Kawazoe & Wakabayashi, 1976; Fukuda et at,, 1984; Matsuka, 1994; Inomata & Matsumoto, 1995,Shir6zu,2006),Itis in foreignillustrated book also written a reference lhat O, noma ranges North America (Higgi n&s Riley ,1970) .However, at presen tthere is no evidence showing that O, norna is disuibute dNorth America includin gAlaska, although there are morphological similarities among noma, bere (tayge taned) potixenes. Although O. taygete and O, bone were treated recently as the same species (Ople r& Wright, 1999; Lukhtanov & Eitschberger, 2001), the relation between O. norna and O. polixene sis un- known, It is ,therefore, of importance to investigat ethe relationship among the Oeneis species. The molecular phylogenic analyses Df the species of genus Oeneis have been car- ried out based on mtDNA (ND5) (Usam iet al., 2005), but only litt lienfbrmatio nis avail- able fbr those based on mtDNA (NDI) ,We have investigat etdhe relationship among noma, polixenes b,ore (tayge taned) melissa through mtDNA NDI (45 2bp) gene analyses. Materials and methods Specimen of O. norna asamana was obtained in the vicinity of Mt Yakushidake ,Japan at al- titude near 2,700 m under the permission of Ministr yof the Environment, Japan (cf *Corresponding author, e-mail: [email protected] .jp NII-Electronic Library Service TThhee LLeepipdiopdteorpoltoegircaollSoocgieitycal Society oofJfap anJapan 294 Takao IToH, Kazuhiko HiRANo and Kazuo wrTANABE Oeneis noma p$ Oeneispolixenes 'M Oeneisbore -$ Fig. 1. 0utline of the distribut iToannges of Oeneis norna. O. potixenes and O, bore. The ranges at Europe were ebtained from Higgin sand Riley (1970 )t,hose in North America were ob- tained from Opler and Wright (1999 )a,nd those in Russia were obtained from Lukhtanov andEitschberger(2001). Acknowledgements) in 2001, Specimen of O. melissa l"cill ahas been obtained in the vicinity of the Loveland pass at altitude near 3,800 m in Rocky Mountains, Colorado ,while one of the authors (T ,Itoh )was staying in Colorado as a visiting Professo rof Universit yof Colorado in 2002. These specimens were dropped into ethanol immediately after capturing. MtDNA NDi sequences of O, polixenes, O. bore (O, ldygete) and e. uhleri as well as Neohipparehia statiiinus used as the reference were obtained from DDBJ data base (Martin 2000), et al., Total genomic DNA was extracted from male specimens, using proteas edigestio nfo11owed by two phenol-chlorofor emxtractions and ethanol precipitation .Mitochondrial gene region of A44DH dehydrogenase subunit 1 (NDI) was applied by PCR using the primers ,5'-CG- TAAAGTCC[[IAGGT[IATATTCAGAI"TCG-3' fOr forward and 5 -ATCAAAAGGAGCTC- NII-Electronic Library Service TThhee LLeepipdiopdteorpoltoegircaollSoocgieitycal Society ooff JJaapapnan Geneticdistances Oeneis 295 among species Table 1.The mt-DNA (NDI) sequences (452 bp) of Oeneis norna, O. potixenes, O. bore and O, metissa. 1 fiO O. 6GGTTATTACAGCCTTTTTCTGATGCTATTAAATTATTTACTAAAGAACA nomaO.polixenes ･･T-･---･･･--･--･-,--････-.･..･..･........,....... ･･T････････････････-･･････････････.･.･･..･........ O.boreO. ･･T･-･･･････････････････････-･････.････..･.･...... melissa 51 100 O. AATTTATTTAAATTATTCAAATTATATTTTTTATTATTTTTCTCCTGTAT norna ･G････CC･G･･･････････-････････････････････.････.･. O. polixenes ･G････････････････.･･･････,･,･･..･.･.....,........ 0.boreO. ･G･･-･C-･-･･････････････････････････････-･････････ metissa 10t I50 O. norna TAAGATTTATGTTATCTTTAATAATTTGGATAGTGATTCCATATTATTTT ･･････････A･･･････････････････････････-･･････,･.･･ O.polixenes ･-････････A･･･････････････････････,･,･･.,,...,.,.. O.boreO. ･-･-･････-A･-････-･･･････････････C･･･C････････････ melissa 151 2oo O. AATTTAATTGTTTTTAATTTAGGGGTATTATTTTTTTTATGTT6TATAAG noma ････････････････････････A･･････････････,･････.･.･･ O.potixenes ･･･････････C･･････A･･･････････････,,･.･..,.....,., O.boreO. tnetissa 201 250 O.norna TTTAGGGGTTTATACTGTTATAATTGCTGGTTGGTCTTCTAATACTAATT ･････････････････････--･･････････A･･･.･.･･..･.･... O. potixenes ････････････････････････････････-A･････,･････,･.･･ O,boreO. ..........,...,........･....･.･･-A･-･･-C･-･･-･････ metisffa 2Sl 300 O. norna ATTCTTTATTAGGGGGATTGCGGGCAGTGGCACAGACTATTTCTTATGAA ･･････････････････････････････････A････.･････.･.･･ O.polixenes ････････････････G･････････････････A･･･････････････ O.boreO. ･･････････････････････････････････A･････-･･A･･････ rrtelissa 301 350 O. noma GTTAGTTTAATTTTATTAATATTATCTAGTATTATTTTAATTTTGGATTT O. pol ixenes .......-G･･･'''''''''''''''''''''''''''''''''''''' O.boreO, melissa 3Sl 400 O. noma T･A･A･T･T･T･A･ATT･･A･A･A･A･T･T･T･A･T･AAG･A･T･T･A･T･C･AAG･G･･A･A･T･T･A･A･T･T･T･G･A･T･T･T･A･T･A.T･T･TA O.potixenes ･･･････T････････････････････A･･････････････････,･･ O. bo reO. ･･･--T---･･-････--･･ ･-････--･-v.･.., metissa 401 45e O. norna TAATATTACCTTTAAGATTTTGTTTTATGTCTTCTTTAATGGCTGAAACT ･-･････････････････A･････････.････..･.･.....･..... O.poiixenes O.boreO. ･--･-･･ T･-････････A･･････････････････････････････ ･･･････････････････A････････････････････A･････････ melissa 45tAA O. noma O,potixenes '' O.bore '' O.melissa Gidfir[[lkGTTTC- f3o'r reverse (Auber tet ai. 1996). Thermal cycle parameter for NDI were 2 min at 940C (1 cycle); 30 s at 94MC, 30 s at 55"C, 1 min at 72"C (30 cycles); 5'min at 720C (1 cycle); 10 min at 4aC (1 ¢ycle) .The PCR product was purifie bdy passing through a gel and fragment sequencing was canied out using a sequence analyzer (ABI Prism 3 1OO). NII-Electronic Library Service TThhee LLeepipdiopdteorpoltoegircaollSoocgieitycal Society ooff JJaapapnan 296 Takao ITeH, Kazuhiko HiRANo and Kazuo WATANABE Table 2, Relative pairwise distance between the Oeneis taxa analyzed using Kimura's twe-parameter rnethod, along with the standard deviation inthe shown parentheses. polixenes bore melissa uhteri O.030(O.O07) O,029(O.O08) (o.oos)o.o7s(o.e13) nornapolixenes O.027 (O,O08)oo..oo223 g(o,oo s)e.o63 (o,o12) boremelissa O,027(O,O08)O.065(O,O13) O.065(O,O13) Results discussion and rlleLb]e In 1, we show the sequences of the partia lmtDNA NDI gene of O, norna and O. melissa, along with the reported sequences of O. polixenes and O. bone (tayget eAm)o.ng mtDNA NDI sequences of the five Oeneis species each consisting of 452 bp, norna dilfers from polixenes by 13 bp, diiier fsrom bore by 13 bp, and differ fsrom melissa by 13 bp, while polixene sdiffer fsrom bone by 12 bp. The habita tof polixene sand bore overlaps in part in North America, and these are treated as distin cstpecies (Howe, 1975). In Europe the habita tof norna and bore also overlaps in part and there bore and noma are treated as dis- tinct species to each other (Higgin &s Ri]ey ,1970) .Outline sof the distributi orannges of norna, polixene sand bore are displaye din Fig. 1. Table 2 shows relative geneti cdistances between the Oeneis taxa analyzed using Kimura's two-parameter method. It is seen in Tbble 2 that there are no appreciable differenc eisn phy- logenic distanc eamong norna, polixenes, bore and melissa, suggesting that ancestors ef these four species have separated at almost the same age. Noteworthy is that the distance between norna and melissa, and that between norna and polixenes are almost the same, al- though norna belongs to the group differe nftrom that of metlisa or bore (Lukhtan o&v Eitschberge r2,001). On the other hand, there is a Iarg egeneti cdistanc ebetween O. uhleri and other four Oeneis species, which is consistent with the fact that the morphology of O. uhleri i' smarkedly differe nitn size and in color from the four other Oeneis species, In 1988, Oeneis philipi inhabitin Aglaska very locall ywas described ,which is morphologi- cally almost indistinguisha blferom polixene s(Troubrid geet al., 1982; Troubridg e& Parshal l1,988) .Further r,ecently O. taygete and O. phitip iwere treated as subspecies of O, bore and O. rosovi, respectively, where tvsevi is an Oeneis species distributin gin the far east edge of Eurasia (Ople &r Wright, 1999; Lukhtanov & Eitschberge r2,001). Lukhtanov and Eitschberger wrote in a recently publishe dmonograph that the male genitali aof O. philipi are identica lto those of O, norna. Therefbre, the conspecificity of O. nonna and O. philipi cannQt be excluded. According to Layberry et al, (1998 O), philipi is conspecific with O. rosovi. Thus, O. rosovi, O. norna and O. philip imay be treated as the same species in future .However, at presen tthere is no clear evidence showing that O. noitna is distrib- uted North America includin gAlaska. This contradicts the descriptio ngsiven in illustrated reference books on Japanese butterfli peusblishe dup to 2006. Acknowledgements and notes The presen twork is based in par ton the survey carried out in 2001 in the vicinity of Mt Yakushi-dake (2,92 6m) locate dat the Northern Japan Alps in Honshu island .The speci- mens have been obtained under the permissio nof Ministry of the Environment, Japan with the permissio ncode, Kan-Chubu-Kyo 280 (2001 )(T .Itoh) ,A part of the presen tresults has been publishe das a DDBJ databas e(AB 1 82583) in 2004. 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