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ISSN 1346-7565 Acta Phytotax. Geobot. 57 (2): i55-164 (2006)

Phylogenetic Analysis ofthe Northern Pinguieula () Based on Internal Transcribed Spacer (ITS) Sequence

KATSUIHIKO KONDOiand HIRO SHIMAI2

iLaboratory qf'Plant Chromosome and Gene Stock Graduate Schoot ofScience, Hiroshima Uhiversity 1-4-3 Klrigamlyama, Higashi-Hiroshima 739-8526 lapan; 27lakamori Orchid Museum, 5i2-73 leuhara, 7bkomori, ?Vdgano 399-3107Jirpan

Internal transcribed spacer (ITS) region of 1 8-26S nuclear ribosomal DNA (nrDNA) in 18 species and three subspecies ofPinguicula distributed in the northern hemisphere was sequenced. Based on thc ITS results, species in Section were basically well-agreed with the orthodox classification. Although three subspecies ofR tongCfblia have been described, the present approach showed them to be separate species, The ITS sequences between R vttigaris and R bohemica were identical and suggested

that the two species could be conspecific as normally treated. Pingtticula aipina did not show any direct relationship with R namosa and R variagata although they have been taxonomically placed in Section Micranthus. [Ilie present ITS study suggestcd that R vittosa, only the speeies of Section Nana, was close- ly related to R ramosa and R variagata. The northern Pinguicula taxa studied here perfbrmed the same seasonal growth pattern, but it was likely as a result of convergent evolution.

Key words: carnivorous , DNA, ITS, Lentibulariaceae, phylogeny, Pinguicttla.

Pinguicttla L. is distributed throughout all continents Fuchs (1982), Zamudio & Rzedowski (199l), Luhrs except fbr Aritarctica and Australasia and is a mem- (1995a) and Blanca et al. (1999). On the other ber of the Lentibulariaceae together with Utricularia hand, in the last few decades, many species of L, and Genlisea St.-Hil. (Juniper et al. 1989, [faylor Pinguicuifa have been described mostly in Europe 1989). Many species of the genus are endemic to (Tammaro & Pace 1987, Zamora et al, 1996, Casper only a locality or relatively small geographical area, & Steiger 2001) and Mexico (Luhrs 1995a, 1995b, and thus, they are often highly endangered (Zamora Speta & Fuchs 1982, 1989, 1992, Zamudio 1988, et al. 1996, 1997), Some species ofthe genus would 1999, 2001). Thus, approximately 90 species are be relict and their distribution have been shrunken hitherto considered valid in the genus. and eyentually migrated to higher elevations of The species of Pinguicula could bc divided mountains or to the north, in the early pestglacialinto three groups (Huxley 1992) on the basis of period (Pennington 1969). physiology, geographical distribution and horticul-

"Nonhern Pinguicula was taxonomically studied by tural points ofview, such as 1) species" or "Temperate Linnaeus (1753), De Candolle (1844), Barnhart sometimes called species;' distributed in (1916) and Ernst (1961). The latest monograph of the mild to colder temperate regions in the northem "Southeastern the genus was completed with three subgenera, 12 hemisphere; 2) North American sections and 46 species by Casper (1966) and some species" mostly distributed in the coastal regions of ` minor modifications of it were proposed by Speta & the southeastern U, S, A,; and 3) ℃ entral American

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TABLE l. Tlie taxa ofthe northern Pingnicula group and other Pinguicula group arid two species of thc outgreup in the Lentibulariaceae studied.ima

Sour-c.g..&. voucherspecimen number

Northern Pinguieuia

Subgenus ll]mnoceras SectionMicranthus RaipinaL, a) Europe to Siberia, and Himalayas (O-4,1OOm), b) Mala Fatra (1,OOOm), Terchova, Slevakia, Kondo & Shimai 5734-LPCGS (HIRO). R ramosa Miyoshi a) E. Japan (1,500-2,300m), b) Koshin-zan (1,700m). Techigi, Japan. Kondo di Shtmai 5735-LPCGS (HIRO). R variegata [[hrcz. a) E. Siberia, Russia, b) Sakhalin Isl., N-49"43'57.S", E-144006'34.9" (240m), Russia. Ik bndo & Shimai 5736-LPCGS (HIRO). Section IVZIna

R villosa L. a) N. Eurepe to Siberia and N. America (O-750m), b) Broad Pass (70em), Cantwell, Alaska, U.S.A. Kbndo & Shimai 5737-LPCGS (HIRO).

Subg¢ nusPinguicula Section Pinguicttla R halcanica Caspcr a) Balkans (1,800-2,400m), VardoUssia b) (1,gOOm), Fokida, Greece. Kbndo & Shimai 5738-LPCGS (HIRO). R hohemica Krajina a) N. Czech Republic (250rn), b) Cesk6 Lfpa (2SOm), Czeeh Republic, Kbndo & Shimai 5739-LPCGS (HIRO). R conyica Bern. et Gren. ex Gren. et Godr.

a) Corsica Isl, (900-2,400m), b) Gorges de la Restonica (1,SOOm), Corsica Isl., France, Kbndo & Shimai 5740-LPCGS (HIRO). R dertosensis (CafiLg.) Mateo et Crespo

a) S to SE. Spain,

b) Perts de Beceit (500m), Tarragona, Spain. Kbndb & S71imai 574 1-LPCGS (HIRO). RfioriiTbmm.etPace a) Maiella (750-2,500m), Italy, b) VaLle dell'Orfento (1,300rn), Abruzzo, Italy, Kbndo & S],imai 5742-LPCGS (HIRO). R gtundlTlora Lam. subsp. grandijiora Lam. a) W. Europc and Morecco (O-2,500m), b) Lac de Fabreges (1,250m), PyrenEes Atlantiques, France. 1(bndo & suimai 701-Cult.LPCGS. R grandijlora Larn. subsp. rosea (Mutel) Casper a) SE. France (650-I,500m), b) Goncelin (650m), Isere, France. Konclo & Shimai 5743-LPCGS (HIRO). R teptoceras Rehb. a) Alps and Apermines (1,1OO-3,OOOm), b) Col de Tende (2,1OOm), Alpes Maritimes, France. Kondo & Shimai 5744-LPCeS (HIRO), R longij?)tia Ram. ex DC. subsp, longijblia Ram. ex DC.

a) Pyrenees (6SO-2,OOOm), Spain, b) Valle de Ordesa (1,500m), Huesca, Spain. Kondo & Shimai 702-Cult,LPCGS,

R longijblia Ram. ex DC. subsp, caussensis Casper

a) MassifCentral (400-7eOm), France, b) Gorges du Ttm (4SOm), Pweyron, France. Kbndb & Shimai 5745-LPCGS (HIRO), R longijblia Rarn. ex DC. subsp, reichenbachiana (Schindler) Casper a) SE. France and Apennines (500-1 ,400rn), b) Vhllee de la Roya (SOOm), Alpes Maritimes, France. Kondo & S7timai S746-LPCGS (HIRO).

maeroceras Link R a) Japan, Sakhalin lsl., Kamchatka to NW/ North America (e-3,OOOm), b) Tbdai-gawa (1,1OOm), Nagano, Japan. Kbndo & Shimai 5747-LPCGS (HIRO).

R mundi Blanea, Jamilena, Ruiz-Rej6n et Zamera

a) Albacete <1,200m), Spain, b) Nacimiento del Rie Mundo (1,200m), Albacete, Spain. Kbndo & Shimai 5748-LPCGS (HIRO).

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TABLE 1. eontinued.

Taxa Seuree & voucher specimen number

R nevadensis (Lindbg.) Casper a) Sierra Nevada (2,400-3,OOOm), Spain, b) Sierra Neyada (3,OOOm), Granada, Spain. Konde & Shimai 5749-LPCGS (HIRO). R potdinii Steiger et Casper a) NE. Italy (500m), b) Nhl d'Arzine (SOOm), Friuli-Venezia Giulia, Italy. Kondb & Shimai 702-Cult.LPCGS.

R vallisneriijbtia Webb a) S. Spain, b) Sierra de Segura (800m), Jaen, Spain. Kondo & Sliimai 5750-LPCGS (HIRO). R vuigaris L, a) N. North America te Eurepe, W. Russia and Morocco (O-2,50em), b) Velka Fatra (500m), Martin, Slovakia. Kbndo & Shimai 5751-LPCGS (HIRO).

Other Pinguicuta Subgenuslsoloba

Sectien lsoloba

P lusitanica L. a) W, Europe and N, Aftica (O-500m), b) Breekcnhurst (30m), Hampshire, England. Kbndo & Shimai 5752-LPCGS (HIRO). SeetienCZirdiop1tyllam

R cr),staUina Sibth. ex Sibth, et Smith

a) Tr6odos (1,OeO-1,800m), Cyprus, b> Tr6edos (1,100m), Cyprus. Kbnde & Shimai 5753-LPCGS (HIRO).

R hirt(flora Ten. a) Italy, Balkans and Turkey (O-2,OOOm), b) Vietri sul Mare (sea !evel), Carnpania, Italy. Kondo & S7iimai 5754-LPCGS (HIRO).

Outgroup

Genlisea violacea St.-Hil.

a) Brazil,

b) Itacambira, Mato Grosso, Brazil. Kbndo & Shimai 703-Cult,LPCGS.

UtriculariaminorL, a) N. hemisphere,

b) Higashi-Hiroshima, Hiroshima, Japan.Kbndo & Shimai 5755-LPCGS (HIRO). Classificationfo11ewedCasper(1966).

a) Generat distribution and altitude range, b) Source of material used in this werk. LPCGS (HIRO)==Vbucher specimen plaeed in the Herbarium ef Hiroshirna University (HIRO). CuLt,LPCGS=Axenic culture strain number ofLaboratory ofPlant Chromoseme and Gene Stock, Graduate Scheol of Science,HiroshimaUniversity.

species" distributed in Mexico to Central America, asexual reproduction (Casper 1962a, 1966, 1972),

but mestly e'ndemic to Mexico. Some exceptional Thus, they have a clear and common seasonal species of the genus grow in the Caribbean and growth pattern in their habitats, They break dor- South America and thus, were non-categorized to mancy and start vegetative growth when the tem- any of the three groups. perature begins to rise in spring and then set , The group of the northern species in Pingui- and finally form hibernacula for dormancy mostly cuia is not necessarily a particular taxonomical by the end of summer. The completion of this rank but could be defined as; 1) it is distributed in growth pattem takes 2-6 months. The majority ofthe

the temperate to arctic or alpine zones in the north- northern species ofthe genus is distributed in Europe

ern hemisphere; 2) it forrns hibernacula to resist and several species are distributed in Asia and North low temperature below OOC and dry season; and 3) America (Casper 1962a, 1966, 1972). It is uncertain it produces gemmae at the base ofhibermaculum for whether or not the similarity in the growth pattern in

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TABLE 2, List efG+C contents and lengths ofthe ITS regiQns ofthe members oftbc northern 1'inguicula studied and their relations

to chromosome numbers.

Basicehromoseme

Len hb G+C% number & Species ITS 1 ITS2 ploid levelChromosome number & authors

R alpina 259 (73.36) 219 (72,60)8(4 × ) 2n=32 Doulat (1947), Sokolovskaja & Strelkova (196e), Casper (l962a, 1966) Rballeanita 254 × (68.5e) 220(69,55) 8(4 )8(8 2n=32 Casper (1966) Rbcthemica 256 (68.75) 218(69.72) × )8(2 2n=:64 Krahulcova & Jaoliova (1991) R carEi'ca 252 (64.68) 218(69.72) × )8(6 2n=16Contandriopoulos(1957) R clettosensis 254 (66.93)218 (69.72) × )8(4 2n= 48 Zamora et al. (1996) Riibith'RgrTaiidi27ana 254 (66,93) 218 (70.18) × ) 2n=32 [[lammaro & Pace (1987)

smbsp. graiidilr7ar:a 254 (65,35) 219 (70.78) 8(4X) 2n= 32 Contaridriopoulos (1 957), Casper (1962a, 1966) RgrTaii{lit7anfi

subsp. rosea 254 (65,35)219 (70.78) 8(4 × )8(4 2n= 32 Contandriopoulos (1 957) Rltlptoeer;as 255 (68.24 218(70.18) × ) 2n[32Contandriopoulos(1957), Casper (1962a, 1966) Rimg[tt7tiZia subsp. ca ussensis 254 (6S.50)218(69.27) n.a. RlongitltiZia

subsp. longmbha 253 × (66,40) 218(71.10) 8(4 ) 2n-32 Casper (r966), ZamoTa et aL (l996) Rlotigifti[i? subsp. reiltbenbathiana 256 (68.75) 218(69,72) 8(4 × ) 2n=32 Contandriopoulos (1957), Casper (1962a, 1966) Rmaatvcevas 254 × (66,93)218(70,18) 8(8 )8(6 2n=64 Uchiyama ( 1 990) Rmtmdi 253 218(70,18) × (65.61) )8(2 2n=48 Zamera et al. (1996) Rnevadmsis 254 (66,14) 218(70,18) × )ll.a.9(22n==16 Zamora et aL (1996) Rpaldbifi' 254 (65.75) 217 (66.36) Rramosa 254 (66.54) 216 (71.30) × )8(4 2n=18Ybshimura(1973) R valltsnendbbZi 254 (68.50)221 (68.78) × )8(8 2n=32 Zamora et al, (1996) R vat:rltga ta 252 (61.11)218 (64,68) × >8(2 2n-64 Zhukeva & Tikhonova (1971) R vdaosa 259 (62.55) 200 (71.00) × ) 2n=16 Doulat (1 947), Casper (1966), LOye & L6ve (1982) R vttigan's 256 (68.75) 218 (69.72) 8(8× ) 2n=64 Casper (1966), L6ve & L6ve (1956, 1982), Doulat (1947)

Lengths (bp) efITS 1 and ITS2 do not include gaps.n.a. = data not available,

the northem species of Pinguicula is due to their (see Tal)le 2). The basic chromosome number ofx=8 genetic similarity or adaptation to their habitat envi- was speculated fbr the taxa studied excepting that of ronment. Casper (1962a, 1966) divided the northem x=9 was expected only in R ramosa Miyoshi Pinguicula into three sections such as Micranthus (2n=18) (Ybshimura 1973). Since their chromo- Casper, IVinna Casper and Pinguicula Casper taking somes were too small sizes, O,5-4,O gm long, to account oftheir morphology and chromosome num- analyze karyotypes (Kondo 1969, YOshimura l973,

bers. Uchiyama i990), further karyomorphological char-

Chromosome numbers ofnorthern Pinguicuin acteristics would net resolve the taxonomical con- species havebeen reported by numerous workers cepts and system ofthe genus.

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Recently, molecular systematics using se- AB 101 fbr forward, and ITS 4 or AB 102 fbr quences of the internal transcribed spacer (ITS) reverse (White et al, 1990, Douzery et al, 1999). region of 18-26S nuclear ribosomal DNA (nrDNA)Basically ITS primers were used, but when the have been developed to evaluate phylogenetic rela- amplification was insuthcient, AB primers were tionships of certain plant groups (Baldwin et al. used. For PCR, sainples with ITS primers were 1995). treated initial 2 min. at 94eC, then, 33 cycles of50

In this paper, the ITS region of18 species and sec. denaturation at 940C, 1 min. annealiRg at 480C

three subspecies of the northern Pinguicula with and 30 sec. extension at 72"C. For AB primers,

three other related species ofthe genus and the out- annealing was at 600C at the same duration as ITS group of Genlisea violacea St,-Hil. and Utricularia primers, For cycle sequencing, PCR products were minor L. in the Lentibulariaceae is studied to ana- purified from collected agarose gel bands using lyze their phylogenetic relationships and the mole- GFX PCR DNA and Gel Band Purification Kit cular results are discussed together with morphol- (Amersharn Biosciences, Piscateway, NJ, U. S, A.), ogy and geograpbical distribution of the taxa. Cycle sequencing conditions regardless of the type

ofprimer were the fo11owing: 96eC for 1 min., then,

Materials and Methods 35 cycles of 10 sec, denaturation at 960C, 5 sec.

aimealing at 50"C, and 80 sec, extension at 72"C. If

The plants used in this study are listed in [[lable 1 , the multiplication was insuMcient, particularly for

Dried leaf materials of the taxa studied were col- hei`barium samples, the cycles were increased up to

lected preferentially from the herbarium specimen or 40. In some cases, DMSO was added for GtC rich

otherwise obtained from tissue culture and cultiva- samples. The ITS regions were sequenced using

tion in Laboratory ofPlant Chromosome and Gene Long-Read Tower DNA sequencer (Amersham

Stock, Graduate School of Sci¢ nce, Hiroshima Biosciences, Piscateway, NJ, U, S, A,)

University, Total DNA was extracted either from

O.025g of dried leaves or from O.07-O,lg of fresh Seguence analysis

leaves ofeach taxon, For related and outgroup taxa, DNA sequences were aligned by Genetyx-Win

Pingnicula crystallina Sibth. ex Sibth. et Smith, R Version 5,2 (Software Development Co., IJJ]D,,

hirtijlora Ten. and R lusitanica L, in the subgenus Tokyo, Japan) and were adjusted manually, The

Isoloba Barnh. and Genlisea violacea and Utr7'culariadata include whole 5,8S and panial sequences of

minor were used. 18S and 26S. Gaps were treated as missing charac-

ters. The sequence data are avai1al)le from the DDBJ

DAC4 isolation, PCR and sequencing databases at the accession numbers ofAB198341 to Samples collected in the process mentioned above AB198365, AB212116 and AB212118. were grinded with liquid nitrogen. Total DNA iso-

1ation from fresh leaves was made with ISOPLANT Phylogenetic analysis rlbkyo, II (Nippon Gene; Japan) fbllowing the man- Phylogenetic analysis was perfbrmed with PAUP* ufacturer's protocol. .DNeasy Plant Mini Kit (Qiagen;4.0 beta 10 for Windows (Sinauer Associates, MA, [Ibkyo, Japan) was used fbr dried leaves. ITS region U. S. A.; SwofTbrd 2002). The mest parsimonious of isolated DNA was ampiified by polymerase chain trees were searched by tree-bisection-reconnection reaction (PCR) using TaKaRa LA 7laqTM (Takaraalgorithrn. The starting tree was obtained via step- Bio Inc,; Shiga, Japan) with GC buffer II. Primers wise addition with random sequence addition.

used fbr the amplification were 20pmol of ITS 5 or Neighbourjoining tree was calculated based on the

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160 APG Nlo1. 57

Distributions

Utricularia minor eutgroup Genlisea violacea ]

R ctystaltina EEE Sect.Cardiophyllum R hirtifiora ]

R tusitanica Sect. Isoloba

R aipinaR E, As variegata As Sect Micranthus R ramosa AsE, ]

R vi"asaR As, Am Sect. Nana

poldnii E

R balcanica E

R valli$neriiiolia E

R longilolia subsp. tongifotia E

R nevadensis E

R corsicaR E

mundiR E

grandMora subsp. gtandifiora E Sect.Anguicula R gtvandifiora subsp. rosea E

R iongifolia subsp. caussensis E

R v"igaris E, Am

R bohemica E

R ieptoceras E

R iongifoLla $ubsp. teiehenbachjana E

R fiOriiR E

ctettosensis E

R mactocetas As, Am

Fv(t. 1 . Phylogenetic tree bascd on ITS sequences ofthe mernbers ofthe northern Pinguicula studied. The number indicates bootstrap '- = = value calculated at 1,OOO replicates, Distribution indicates approximate distribution rangcs (E Europe, As Asia and Am North America); for mere detailed distribution, see Ihblc 1, Dot linc indicates that the ITS sequence between the two is identical. Plant llames indicated in a box are nonhern Pinguicula. C]assification fo11ewed Casper (1966).

Kimura's two-parameter distance. Bootstrap test Godr. and R variegata Tur¢ zaninov, The length of

was perfbrmed with 1,OOO replicates for the maxi- 5.8S was 163 bp, a uniformed size among all ofthe mum parsimony analysis. The phylogenetic tree taxa examined here. The longest ITS 2 was 221 bp was edited by TREEVIEW Version 1 . 6, 6 (UniveTt-in R vallisneriij2)lia Webb, and the shortest ITS 2 sity ofGlasgow, Scotland). was 200 bp in R viilosa. The lengths ofITS 1 and 2

in the related taxa and the outgroups were listed as

Results and Discussion fbllows: 258 bp of ITS 1 and 219 bp oflTS 2 in R

lusitanica, 242 bp of ITS 1 and 221 bp ofITS 2 in P

The total ]ength ofboth ITS 1 and ITS 2 including cr]rgtallina, 241 bp ofITS 1 and 220 bp ofITS 2 in

5,8S nrDNA, but excluding gaps, in the northern R hirtij7b,u, 254 bp ofITS 1 and 230 bp ofITS 2 in Pingtticula varied. The length ofITS 1 in the nomh- the outgroup Genlisea violacea and 162 bp oflTS 1

em Pinguicula varied from 252-259 base pairs fop) and 228 bp of ITS 2 in the other outgroup

and that ofITS 2 varied from 200-221 bp, excluding Ulrricularia minor,

gaps (Ibble 2), The longest ITS 1 was 259 bp in R The percentage of G+C in both ITS 1 and ITS

aipina L, and R villosa L., while the shortest ITS 1 2 of the northern Pinguicula was comparatively

was 252 bp in R corsica Bern. et Gren, ex Gren. et higher than the other species reported by Baldwin et

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August 2006 KONDO & SHIMAI: Phylegeny in nemhern Pinguicuta 161

al. (1995). In the northern Pinguicula group, the separatc species as Mateo & Crespo (1995) and highest G+C content was 73,36% in ITS 1 and Zamora et aL (1996) treated. The ITS sequences 72.60% ofITS 2 both in R alpina, while the lowest betwecn Il bohemica Krajina and R vuigaris L.

G+C content was 61.1 1`}6 in ITS 1 and 64.68 in ITS were idcntical and implied that R bohemica could be

2 both in R variagata, The percentage ofG+C in a Bohemian local form ofR vuigaris as normally Sect. Pinguicula were between 64.68 and 68,75% in considered (Krahulcova & Jarol{mova 1 991 ). ITS 1 and between 66.36 and 71,10% in ITS 2. Pinguicula ramosa, R variagata and R vil- The percentage of G+C in Sect. Cardiophyllum losa would be phylogenetically closely related to

Casper was 57,02% in ITS 1 and 57,Ol% in ITS 2 each other, Pinguicula ramosa was censidered a

studied in R crystallina and 56.029t6 in ITS 1 and synonym ofR variegata due to morphological sim-

56,82% in ITS 2 studied in R hirtij7ora, ilarity according to Ernst (1961). The morphologi- cal close relatien of R ramosa and R villesa was

PIrylogerry of the northern Pinguicula suggested by Tamura (1953). However, R ramosa The ITS result suggested that the northem Pin- showed the chromosome number of 2n=18

guicula studied could be divided into three groups; (Ybshimura 1973) that was different from that of the first group ofonly R aipina ofsect, Mictunthus, 2n=64 in R varic gata (Zhukova & Tikhonova 1971) the second group of R vamosa and R variagata of and that of2n=16 in R villosa (Casper 1966, L6ve sect. Micranthus and R villosa ofsect. Ndna, and & L6ve 1982) ([Rible 2). The present ITS result third group of all the species of sect. Pinguicula supported that R ramosa was difTk)rent frem R var-

studied. The data showed disagreements with the iegata but was more closely related to R villosa.

division of sect. vacranthus and sect. Ntina, pro- According to the ITS phylogeny, the evolu- posed by Casper (1966). However, the ITS result tionary relationship ofR alpina might be different supported the sectional division of sect. Pinguicula from that of the othcr species of the northern

based on the morpholegical classification proposed Pinguicula as it divided at the base ofthe clade of by Casper (1966) although polygamy was seen in the other northern Pincuicula with higher bootstrap sect. Pinguicula (Fig, 1), value, Although the geogTaphical distribution of Three subspecies ofR longijbtia Ram, ex DC. the taxonomi ¢ ally closely related R lusitanica of

have been taxonomically recognized, but they did sect. lsoloha Casper and R cr ystatlina and R hirti- not form a branch group based on the ITS result ]7bra ofsect. CantiopIrytlttm were sympatric in part suggesting that they could be different species as with that ofthe nonhern Pingtiicttla species, they did

sometimes suggested (Caspcr 1959, Degljareva et al. not form any clade with any specics ofthe northern 2004). Funher investigations with mu]tjple methods Pingtticula studied in the present molecular analy- are necessary to justify the taxonomy of the three sis. At least, R cr:ystallina and R hirtCflbra were subspecies ofR longijblia, Pinguicula dertosensis likely more primitive than the other Pinguicula (Cafiig.) Matee et Crespo has been taxonomically species studied.

very controversial and was initially considered con-

specific with R grandij7ora Lam. (Cafiigueral 1957, Moiphology of the species ofthe northern Pingui-

Bolbs & Vigo 1 983), but more recently it was treat- culastudied

ed as a subspe ¢ ies ofR longijZ)lia (Schlauer 1994) Most ofthe specjes in the northern Pinguicula has and furthermore as a distinct species (Mateo & leaves with the cornmon characters of oblong-lance- Crespo 1995, Zamora et al. 1996 as R submediter- olate te ovoid blades with petiole. Several leaves

ranea), Our own ITS approach supported them to be form a rosette on the ground. Without flowers it is

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162 APG Vbl, S7

often diMcult to identify these species. In ¢ ontrast, tvata H, B, K. and R chilensis Clos, both in sect,

Pinguicula long(folia subsp. Iongijblia, subsp. Anu)ullipakitum Casper, but rather uncommon in the reichenbachiana (Schindler) Casper, ll mundi genus. It might support that R aipina would be Blanca, Jamilena, Ruiz-Rej6n et Zafnora and R phylogenetically isolated from the other northern valiisneriijblia possessed oblong to linear leaves Pinguicula species, as discussed above. up to 1O-35 cm long, that showed a clearly difTerent Among 21 taxa of the northern Pinguicula

appearance compared with the other species ofthe studied, only R aipina, R maeJvceras, R vuigaris

northem Pinguicula. The leaves ofR long4foiia andR viliosa were widely distributed in the north-

subsp, longijblia, subsp, reichanbachiana and R ern hemisphere; many taxa in sect. Pinguicula were

vallisneriijblia were particularly erect at the base and endemic to certain small areas in Europe, and R pendent toward the tip. Howeyeg they were also difi ramosa and R variegata in sect, Mieranthus were

ficult to identify each ofthose taxa without . endernic to a certain small area or very sparse in

Pinguicula longtfolia, R mundi and R vaUisneriijblia East Asia (Casper 1966), were combined together to be placed in sect, The branch group ofR variegata, R ramosa Longij2)liae (Casper) Blanca, Ruiz-Rej6n et Zamora and R viUosa could be East-Asiatic although A vii- according to morphological similarity by Blanca losa was a circumpolar species (Fig. 1, Tbble 1). It et al. (1999). However, the present ITS result could was unclear whether or not this branch group was not resolve those relationships. On the other hand, R originated from a European species with a rela-

namosa, R variegata and R villosa phylogenetical- tionship with sect. Pinguicttla during the course of

ly so closely related as discussed above had com- this investigation.

monly much smaller leaves ca. 1 cm long than the Regarding the present ITS result, R balbanica

other species of the northern Pinguicuta had and and R vallisneriijblia, fbrrning the same branch

forrned a branch group in the ITS result with rela- group, geographically jso]ated from each other and

tively higher bootstrap value at 80 and 88%, the same tendency was seen in R ,fiorii, R der- Taxonomy of the species of the northern tosensis and P macroce]rts (Fig, 1, [Ibble 1), idmother Pinsuicula is regarding the charaeteristics ofleaves branch group from R' longijblia subsp. iongijblia to

and flowers. However, certain species efthe north- R grandij7ora subsp. rosea mostly occurred in

ern Pinguicula have very similar flowers in mor- France or more west, while that from R iongijblia

phology with each other which make their identifi- subsp. caussensis to R longijblia subsp. reichen- cation confused. For instance, il macroceras Link bachiana occurred in France or more east (Fig. 1, "lable and R vuigaris were quite dithcult to separate and 1). The ITS tree implied that the rnember of are often considered conspecific (Casper 1962b), Sect, Pinguicula might be originated from Europe Then, two specjes were carefu11y, quantitativelyand expanded to East Asia and North America. observed with respect to fiower and were conclud- [[he northern Pingtticula taxa studied here per- ed separate species by Casper (1 962b). Despite the formed the same seasonal growth pattern, but it fioral similarity, the present ITS study determined was likely as a result of convergent evolution. that the two species were likely separate species. Further molecular phylogenetic irwestigation should

Roots ofthe species ofthe northern Pinguicula be expected in the throughout sections ofPinguic"la were usually short, thin and seasonal growth, except- to clarify andjustify our taxonomical concept of the ing the roots ofR aipina were long, thick and non- genus.

seasonal. Such a fibrous elastic root system ofR

aipina was also seen in South American R ca4zp- The authors wish to cxpress their sincere thanks to Dr,

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August2006 KONDO & SHtMAI/ Phylogeny in northern Pinguicula 163

Tbrnohisa Yinkawa and Mrs. Ibmoko Fu.iimoto, Tsukuba (Friuli-Vit)nczia Giulia)] Pinguicula poldinii Steiger Botanical Garden, National Science Museum fbr techni- et Casper spec. nov, Wulfenia 8i 27-37. cal assistance and helpfu1 suggestions for ITS data col- Contandriopoulos, J, 1962, Recherches sur la flore lections and analyses, Thanks are alse due to Prof Dr, endemique de Ia Corse et sur ses origines, Ann. Sadashi Komiya, Nippon Denta1 College, Dr. Lubomir Facult. Sci. Marscille 32: 1-354,

Adamec, Teebefi, Czech Rcpublic and Mr. Kamil Pasek, De Candolle, A. P, l844. Prodromus System, Natur. Reg.

Best Camivorous Plants, Czech Republic for supplying Veget. 81; 26-32,

materials. This study was supported by the Grant-in-Aid Degljareva, G., J. Casper, F. Hellwig & D. Sokolotf.

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RecelvedJiznua,:y 6, 2005; accepted Flebruaty 8, 2e06

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