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タイトル CHROMOSOMAL CYTOLOGY AND EVOLUTION IN Title () Watanabe, Kuniaki / King, Robert M. / Yahara, Tetsukazu 著者 / Ito, Motoni / Yokoyama, Jun / Suzuki, Takeshi / Author(s) Crawford, Daniel J. 掲載誌・巻号・ページ Annals of the Missouri Botanical Garden,82(4):581-592 Citation 刊行日 1995 Issue date 資源タイプ Journal Article / 学術雑誌論文 Resource Type 版区分 publisher Resource Version 権利 Rights DOI 10.2307/2399838 URL http://www.lib.kobe-u.ac.jp/handle_kernel/90002998

Create Date: 2017-12-18 CHROMOSOMAL CYTOLOGY Kuniaki Watanabe,2 RobertM. King,3 Ito,5 AND EVOLUTION IN JunTetsukazu Yokoyama, Yahara,4 Takeshi Motoni Suzuki,7 EUPATORIEAE and Daniel J. Crawford" (ASTERACEAE)'

ABSTRACT

Reportsof 68 new chromosomecounts attributed to 53 species from25 generaof Eupatorieaeof the Asteraceae, based mostlyon determinationsof mitoticmaterials, include first counts for 2 genera(Acanthostyles and Lepidesmia) and 14 species and new reportsfor 8 species. B chromosomesare reportedfor 4 generaand 12 species. Karyotype analysesmade on 20 species of Eupatorieaeand one species of Heliantheaeshowed that total karyotypic lengths of the taxa withn = 16-19 of helianthoidand eupatorioidtaxa are comparableto thoseof some eupatorioidtaxa withn = 10. This is contraryto the previoushypothesis that the higherchromosome numbers n = 16-19 werederived from n = 10 by polyploidizationfollowed by dysploidloss. Cytologicaldata supplementand are consistentwith the following conclusionspredicted from molecular phylogenetical and biochemicaldata: (1) The ultimatebase numberof Eupatorieae is 17, and thelower numbers are derivedby successivedysploid reductions; (2) A reductionin chromosomaland total karyotypiclength accompanied by evolutionaryadvancement has been revealedfor some generaand species within this tribe;(3) A high base numberof x = 17 in Eupatorieaeis consideredto be deriveddirectly from one of the membersof Heliantheaewith n = 17 to 19.

The Eupatorieaeare a verydiversified tribe and chromosomenumbers are amongthe mostreadily consistof morethan 2300 species and 180 genera definedtaxonomically. Therefore, we attemptedto in 18 subtribes(King & Robinson,1987). In spite find clues regardingphylogenetic relationships of its representinga large elementof Asteraceae, throughoutthe tribe by assessing chromosomal the tribeEupatorieae has remainedpoorly under- variation,restriction site mutationsin chloroplast stoodbecause its centersof diversity are in Central DNA (Ito et al., in prep.),and the utilityof isozyme Americaand ,which were remote numberfor determining ploidy level (Suzukiet al., fromthe early centers of study. Recent monograph- unpublished). ical (King & Robinson,1987), cladistical (Karis, Our purposesin thispaper are to reportoriginal 1993; Bremer,1987, 1994; Bremeret al., 1992), countsof chromosomenumbers in Eupatorieae,to and molecularphylogenetic works (Jansen et al., comparethem with numbers reported previously, to 1991; Watsonet al., 1991; Kim et al., 1992) have analyze karyotypesof selected representativespe- stimulatedus to studythe originand evolutionof cies, and to apply the resultsto a betterunder- Eupatorieae.A broad range of base chromosome standingof the origin, phylogeny, and chromosomal numbers,from n = 4 to 25, has been reported(Fig. evolutionin the tribe. 1). The remarkableconstancy of chromosomemor- MATERIALS AND METHODS phologyand numberwithin genera has been in- valuable in the studyof systematics.In ad- Cytologicalobservations were made primarilyon dition,many of the groupsthat have distinctive mitoticcells of roottips obtainedfrom seedlings

'We thankThomas B. Croatof the MissouriBotanical Garden in St. Louis, Missouri,Gerald R. Carrof the University of Hawaii,J. Rzedowskiof the Institutode Ecologfain ,and David B. Lellingerof the SmithsonianInstitution in Washington,D.C., forcollecting viable seeds forthis study.We also thankVictor Castro of the Universityof Costa Rica, San Jose,for his help in collectingplant material in Costa Rica and Harold Robinsonfor his help withidenti- fications.This studyis supportedby a Grantin Aid forOverseas Researchfrom the Ministryof Education,Culture and ScientificResearch, Japan, No. 03041028. 2 Departmentof Biology,Faculty of Science, Kobe University,Tsurukabuto 1-2-1, Kobe, 657, Japan. 3Department of Botany,National Museum of NaturalHistory, Smithsonian Institution, Washington, D.C. 20560, U.S.A. 4Department of Biology,Faculty of Science, KyushuUniversity, Fukuoka, 812, Japan. 5Department of Biology,Faculty of Science, Chiba University,Yayoi-cho, Inage-ku, Chiba, 260, Japan. 6 BotanicalGardens, University of Tokyo, Hakusan, Bunkyo-ku, Tokyo, 112, Japan. 7Museum of Natureand HumanActivities, Hyogo, 6 Yayoigaoka,Sanda, 669-13, Japan. 8 Departmentof Plant Biology,Ohio StateUniversity, Columbus, Ohio 54321, U.S.A. ANN. MISSOURi BOT. GARD. 82: 581-592. 1995. 582 Annals of the MissouriBotanical Garden

15 somaticchromosomes of eightrepresentative spe- Basic chromosome number In Eupatorleae cies. Chromosomenumber determinationsfrom 68

.0 populationsattributed to 53 species from25 genera of Eupatorieaeare reported.First reports are given forAcanthostyles buniifolius (2n = 20), Adenostem- 0 ma cuatrecasalsii(2n = 20), AgeratinaisoleJ)is (2n

E2 34), Decachaeta thieleana (2n = 32), Fleisch- z mannia sideritides(n = 10, 2n = 20), Lepidesmia squarrosa(2n = 20), Koanophyllonlongifioium (2n 30), Mikaniacongesta (2n = 34+0 to 3B), Neo- mirandeaarthodes (n = 17+0 to 5B), N. guevarii (2n = 50+2 to 12B), N. parasitica(2n = 34+1 to 2B), N. standleyi(2n = 50+1 to 4B), Stevia con- nata (2n = 44), and S. suaveolens(2n = 33+0 to 2B), and new reportsare givenfor roth- 50 rockii(2n = 51 and 68), Chromo/aenalaevigata (2n = 50), Mikania scandens(2n = 34 + 0 to I B), Praxelisclematidea (2n = 30), Steviaeupatoria (2n = 36 and 48), S. monardaefilia(2n = 33). S. pilosa (2n = 22), and S. tomentosa(2n = 33). Remaining countsconfirm the chromosome numbers of all pre- viousreports in somespecies or are consistentwith one ofvariable numbers reported by several authors 0 in otherspecies. Of the 68 accessions in which chromosomenumber determinations were made, 53 I} I (78%) are diploids and 15 (22%) are polyploids. The presence of B chromosomesis reportedfor

0 100| Bartlettinasordida, Mikania congesta, M. scandens, Neomirandeaangularis, N. arthodes,N. bIifora,N. guevarii,N. parasitica,N. standleyi,Stevia salici- folia, S. serrata,an(1 S. suaveolens.The B chro- mosomes are distinctlysmaller than autosomies (Fig. 3), except in Mikania an(1 Stevia,anr( show 4 9 10 11 12 15 16i17 15 19 20 25 Baslc chromosome number the early con(lensationtypical of the centromieric regionsof autosomes at prometaphase.Both Mikan- FIGURE 1. Distributionsof base chromosome numbers ia and Steviahave severalsmall autosomes in the species, genera,and subtribesof Eupatorieae. nearly the same size as B chromosomes(Fig. 7). grown from fruits of known provenance in the greenhouse of Kobe University.For the cultivation KARYOTYPE of specimens and the preparation of materials for Figures10-30 are haploidkaryotypic idiogramns. cytological work, the procedures of Watanabe et al. Drawingsare based on the means of ten measure- (1975) and Watanabe et al. (1990) were followed. mentsfor each. Each idiogramis arrangedin de- The measurements of karyotypiclength were made scendingorder of chromosomenumber within Eu- on root tip mitoses, and each measurement is the patorieae next to helianthoil Podachaenium mean of measurements of five metaphase plates. eminens(Lag.) Sch. Bip. Voucher specimens have been deposited in US and MAK. In Table 2, we reportthe mean totalkaryotypic lengthswith their standard errors, the rangesfrom RESULTS the longestto shortestchromosome amidi their ratio foreach karyotype,arid the mean arm ratio(total CHROMOSOME NUMBER long arm length/totalshort arm length).The two All chromosome number determinationsare pre- lattermeasurements indicate the degreeof karyo- sented in Table 1. Figures 2-9 are photographs of typic asymmetry.Significance of differencesbe- Volume 82, Number4 Watanabe et al. 583 1995 ChromosomalCytology in Eupatorieae

Table 1. Chromosomenumber determinations in Eupatorieae(Asteraceae). n countsare frompollen mothercells (PMCs) and 2n countsare fromroot tips.

Chromosomenumber Species n 2n Collectiondata or reference Acanthostylesbuniifolius (Hook. & 20 Argentina.Buenos Aires:Parana Delta at Ti- Arn.)R. M. King & H. Rob. gre,King 10288 (US) Adenostemmacuatrecasasii R. M. 20 .Merida: 4.1-6.3 kmE of Santo King & H. Rob. Domingo,King 10538 (US) A. viscosumJ. R. & G. Forst. 20 U.S.A. Hawaii: KawaihauDist., Makaleha Mtns.,SSW of Puu Eu, Flynn5461 (US) Ageratinaanisochroma (Klatt) R. 34 Costa Rica. 10 km W of San Ramon,15 M. King & H. Rob. Aug. 1991, Yaharaet al. s.n. (MAK) A. bustamenta(DC.) R. M. King & 1711 -Costa Rica. 27 km N of San Isidro,Yahara H. Rob. et al. 39 (MAK) A. havanensisR. M. King & H. 34 Mexico.: near the Universityin Rob. Saltillo,King 10379 (US) A. herbacea(A. Gray)R. M. King 34 U.S.A. : BrewsterCo., Chisos Mts.,Mt. & H. Rob. Emory,Yahara et al. 13 (MAK) A. isolepis(B. Rob.) R. M. King & 34 Mexico.: 13 km fromPachuca to El H. Rob. Chico on Hwy.105, 17 Oct. 1993, Yahara & Ito s.n. (MAK) A. rothrockii(A. Gray)R. M. King 51 U.S.A. Texas: JeffDavis Co., Old FortDavis, & H. Rob. Yaharaet al. 7 (MAK) 68 U.S.A. Texas: BrewsterCo., Chisos Mts.,Mt. Emory,9 Aug. 1991, Yaharaet al. s.n. (MAK) A. scorodonioides(A. Gray)R. M. 34 Mexico.Coahuila: 2 km E of Los Lirios, King & H. Rob. King 10326 (US) A. wrightii(A. Gray)R. M. King & 34 U.S.A. Texas: BrewsterCo., Chisos Mts.,Mt. H. Rob. Emory,Yahara et al. 14 (MAK) Ageratumcorymbosum Zuccagni 40 Mexico.Coahuila: 3 km S of Saltillo,King 10368 (US) A. microcarpum(Benth. in Orsted) 1011 Costa Rica. 15 kmS of Cartago,King 10236 Hemsl. (US) 1011 Costa Rica. 27 km N of San Isidro,King 10241 (US) 1011 Costa Rica. VolcanIrazu, 16 Aug. 1991, Ya- hara et al. s.n. (MAK) 1011 Costa Rica. 0.5 km NE fromMt. Orosi,17 Aug. 1991, Yaharaet al. s.n. (MAK) 1011 20 Costa Rica. The summitof Mt. Orosi,17 Aug. 1991, Yaharaet al. s.n. (MAK) Austroeupatoriuminulaefolium 20 Argentina.Buenos Aires:city limit of Bue- (HBK) R. M. King & H. Rob. nos Aires at the municipaltrain station, King 10282 (US) Bartlettinasordida (Less.) R. M. 1611 Australia.Victoria: cultivated in Royal Bo- King & H. Rob. tanic Gardens,King s.n. (US) 32 + lB Australia.Victoria: cultivated in Royal Bo- tanic Gardens,Watanabe s.n. (MAK) Brickelliabetonicifolia A. Gray 18 U.S.A. Texas: JeffDavis Co., Old FortDavis, Yahara et al. 8 (MAK) B. chlorolepis(Wooten & Standl.) 18 Mexico.Coahuila: ca 9 kmfrom Los Lirios, Shinners King 10320 (US) B. grandiflora(Hook.) Nutt. 18 U.S.A. Texas: BrewsterCo., Chisos Mts.,Mt. Emory,9 Aug. 1991, Yaharaet at. (MAK) Carminatiatenu flora DC. -20) U.S.A. Texas: JeffDavis Co., Davis Mts.,Ya- hara et at. 4 (MAK) 584 Annals of the MissouriBotanical Garden

Table 1. Continued.

Chromosomenumber Species n 2n Collectiondata or reference Carphochaetebigelovii A. Gray 22 U.S.A. Texas: BrewsterCo., Chisos Mts.,Mt. Emory,9 Aug. 1991, Yaharaet al. s.n. (MAK) Chromolaenalaevigata (Lam.) R. 50 Argentina.Buenos Aires:Parana Delta at Ti- M. King & H. Rob. gre,King 10287 (US) Conocliniumgregii (A. Gray)Small 20 U.S.A. Texas: BrewsterCo., Chisos Mts.,Mt. Emory,Yahara et al. 15 (MAK) Decachaeta thieleana(Klatt. ex T 32 Costa Rica. San Jose:San Ramonde Tres Durand& Pittier)R. M. King & Rios, Nov. 1993, Castros.n. (US) H. Rob. Fleischmanniamicrostemon (Cass.) 411 Costa Rica. 3 km S of Santa Cruz,King R. M. King & H. Rob. 10243 (US) 411 Costa Rica. 0.5 km NE and on the footof Mt. Orosi,17 Aug. 1991, Yaharaet al. s.n. (MAK) F sideritides(Benth. in Orsted)R. 10l 20 Costa Rica. TapantiNational Wildlife Ref- M. King & H. Rob. uge, Yahara et al. 128 (MAK) & King 10262 (US) Hebecliniummacrophyllum (L.) DC. 20 Trinidad& Tobago.St. George:along North Coast Road, 5 km W of Maracas Bay Vil- lage, Wasshausen1811 (US) Isocarphaatriplicifolia (L.) R. Br. 20 Costa Rica. Santa Rosa N. P., 15 Aug. 1991, ex DC. Yaharaet al. s.n. (MAK) Koanophyllonlongifolium (B. Rob- 30 Mexico.Coahuila/Nuevo Leon: just E of La- inson)R. M. King & H. Rob. guna de Sanchez,King 10341 (US) Lepidesmiasquarrosa Klatt. 20 Venezuela.Falcon: ca. 80 km S of Coro, King 10270 (US) Liatrisspicata (L.) Willd. 20 U.S.A. NorthCarolina: Spring Lake, 7 Oct. 1991, Ito et al. s.n. (MAK) Microspermumdebile Benth. 24 Mexico.Oaxaca: 47.7 kmfrom Guelatao, 30 Sep. 1993, Yahara & Ito 231 (MAK) var.arsenei Benth. 24 Mexico.Michoacan: near Patzcuaro,Medina 2610 (US) Mikania congestaDC. 34 + 0 - 3B Argentina.Buenos Aires:city of Buenos Ai- res, banks of the Rio de La Plata, King 10285 (US) .A. scandensL. Willd 34 + 0 1Bl U.S.A. NorthCarolina: Spring Lake, 6 Oct. 1991, Ito et al. s.n. (TI) Neomirandeaangularis (B. L. 50 Costa Rica. Cartago:Volcan Turrialba, King Rob.) R. M. King & H. Rob. 9695 (US) 50 + lB Costa Rica. San Jose:La Lucha, 27 July 1987, Lellingers.n. (US) 2511 Costa Rica. 15 km S of Cartago,Yahara et al. 22 (MAK) & King 10238 (US) 2511 Costa Rica. 0.5 km NE fromMt. Orosi,Ya- hara et al. 66 (MAK) & King 10244 (US) 2511 Costa Rica. Near the summitof Mt. Orosi, King 10246 (US) N. arthodes(B. L. Rob.) R. M. 1711+ 0 - Costa Rica. Cartagoto San Isidro,King King & H. Rob. 5B 10239 (US) 1711+ 0 - Costa Rica. Cartago:Tapanti National Wild- 2B lifeRefuge, King 10261 (US) N. bifloraR. M. King & H. Rob. 50 + 2 - 6B Costa Rica. VolcanTurrialba, Yahara et al. 90 (MAK) & King 10251 (US) N. guevariiR. M. King & H. Rob. -50 + 12B Costa Rica. Cartago:ca. 7 kmfrom the dam at Tapanti,King 9674 (US) Volume 82, Number4 Watanabe et al. 585 1995 ChromosomalCytology in Eupatorieae

Table 1. Continued.

Chromosomenumber Species n 2n Collectiondata or reference 50 + 2 - 6B Costa Rica. Cartago:Tapanti National wild- lifeRefuge, King 10260 (US) N. parasitica(Klatt) R. M. King & 34 + 1 - 2B Costa Rica. 8.9 mi. NW of centersof Sanra- H. Rob. min,Croat 68077 (US) N. standleyi(B. L. Rob.) R. M. 50 + 1 - 4B Costa Rica. La Hondura,Bruallio Carrillo N. King & H. Rob. P., King 10263 (US) Polyanthinanemorosa (Klatt) R. M. 1011 Costa Rica. On the wayfrom Cartago to Ta- King & H. Rob. panti,W limitof Paraiso,King 10247 (US) Praxelisclematidea (Griseb.) R. M. - 30 . Rio de Janeiro:City limit of Rio de King & H. Rob. Janeiro,King 10281 (US) Sclerolepisuniflora (Walter) Britten, 30 U.S.A. Delaware:Sussex Co. W of Bethamy Sterns& Poggenb. Beach, King 10235 (US) Steviaconnata Lag. 44 Mexico.Oaxaca: 4.9 km fromEl Punto.Ya- hara & Ito 142 (MAK) S. eupatoria(Spreng.) Willd. 36 Mexico.80 km fromMexico City to Tulan- cingo,Yahara & Oyama 414 (MAK) 48 Mexico.Hidalgo: 11 kmfrom entrance to El Chico, Yahara & Oyama 451 (MAK) S. microchaetaSch. Bip. 24 Mexico.Oaxaca: 54.3 km N fromGuelatao, Yahara & Ito 251 (MAK) S. monardaefoliaHBK 33 Mexico.: 4 km N fromState border, Ixta-PopoN. P., Yahara & Ito 381 (MAK) S. ovata Willd. 33 U.S.A. Texas: BrewsterCo., Chisos Mts. Mt. Emory,9 Aug. 1991, Yaharaet al. s.n. (MAK) S. pilosa Lag. 22 Mexico.Hidalgo: 10 kmfrom Pachuca to El Chico on Hwy.105, Yahara & Ito 425 (MAK) S. salicifoliaCav. 24 + 0 lB Mexico.Coahuila: 2 km E of Los Lirios, King 10327 (US) S. serrataCav. 33 + 0- 2B Mexico.80 kmfrom Mexico City to Tulan- cingo,Yahara & Oyama 412 (MAK) S. suaveolensLag. 33 + 0- 2B Mexico.80 kmfrom Mexico City to Tulan- cingo,16 Oct. 1993, Yahara & Oyama s.n. (MAK) S. tomentosaHBK 33 Mexico.80 kmfrom Mexico City to Tulan- cingo,Yahara & Oyama 411 (MAK) 33 Mexico.Hidalgo: 10 kmfrom Pachuca to El Chico on Hwy.105, Yahara & Oyama 422 (MAK)

tweenmean total karyotypic lengths were evaluated (t = 9.976; p < 0.001) withthe same chromosome by a t-test. number,2n = 34 (Table2 and Figs. 4-7). Similarly, Althoughthere is some variationin the total thoseof Koanophyllon longifolium (t = 5.027; p < karyotypiclengths and chromosomallengths be- 0.01), Lepidesmiasquarrosa (t = 3.851; p < 0.02), tween and withinspecies, there is a significant and Fleischmanniasideritides (t = 5.868; p < lengthdifference between genera withinEupato- 0.005) have also been shown to be significantly rieae. The chromosomaland totalkaryotypic length shorter(compared with the totalkaryotypic length ofMikania congestahave been shownto be signif- in Hebecliniummacrophyllum) than those of other icantlyshorter than those of Neomirandeaparasi- species and genera with the same chromosome tica (t = 11.137; p < 0.001), Ageratinascorodon- number,2n = 20 (Figs. 8, 9). The chromosomal ioides (t = 10.943; p < 0.001), and A. havanensis and totalkaryotypic lengths of Carphochaetebige- 586 Annalsof te Missouri Botanical Garden

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FIGuRES 2-5. Photomicrographsof somatic metaphase chromosomes.-2. Podachoenium eminens (2n = 38).-3. Neonirandeaguevarii (2n = 50+ 12B).-4. Ageratiuascorodonioides (2n = 34).-5. Barkttuinasdida (2n = 32+ IB). Scale bar = 5 &m. Arrowheads indicateB chromosomes.

107iiwith 2n = 22 are the longestamong the taxa of Ageratinascorodonuoides (t = 2.189; 0.10 > p examinedin thistribe. > 0.05) with2n = 34, as well as to thatof Bart- Total karyotypiclength of Podachaeniumermu- lettinasordida with 2n = 32 (t = 0.374; p > 0.50). nens (2n = 38, Heliantheae)is comparableto that In addition,total karyotypic lengths of those spe- Volume 82, Number4 Watanabe et al. 587 1995 ChromosmnalCytology in Eupatoreae

44,

%S~ t. 'etC*:

8 1 9 a FIGuRES6-9. Photomicrographsofsomatic metaphase chromosomes.-6. Neomirandea parasitic (2n = 34+1B). 7. Maikaniacongesta (2n = 34+3B).-8. Eupatoriwmpurpureum (2n = 20).-9. Fleischmanniasideriides (2n = 20). Scale bar= 5 pm.Arrow heads indicate B chromosomes. cies with higherchromosome numbers are also fromprevious reports, Table 3 shows the revised comparable (compared with the total karyotypic distributionof base chromosomenumbers in Eu- lengthin Ageratinahavanensis) to those of Aus- patorieaeaccording to thesubtribes of King & Rob- troeupatoriuminulaefolium (t = 1.212; 0.40 > p inson (1987); underlinedbase chromosme num- > 0.20) and Acanthostylesbunuifolius (t = 1.703; bers wereconfirmed in the presentstudy. We have 0.20 > p > 0.10). with2n = 20. In contrast,the not yet determinedchromosome numbers for Hof- differencebetween the total karyotypiclength of meisterunaeand Oaxacaniinae ourselves,and we species with2n = 50 and 34 withinthe genus Neo- are not certainif the base numberx = 18 is cor- murandeais significant(t = 4.399; p < 0.02). rect. The overalldistribution of chromosome numbers DISCUSSION in Eupatorieaeis characterizedby havingfew spe- Althoughprevious reports for chromosome num- cies and generawith numbers smaller than n = 10, bers have includedmany miscounts or have been comparedto all othertribes in Asteraceaeexcept based on misidentifiedplant materials,chromo- Barnadesieae,Mutisieae, and Cynareae.Also, Eu- some numbers,including the presentdata, are now patorieaeis unusualbecause thelower chromosome knownfor over 467 species in 96 generaof all 18 numbersare completelylacking in six of the sub- subtribesof Eupatonreae(compiled from the index- tribes,and the occurrencesof B chromosomesare es to chromosomenumbers for 1958-1991: Fedo- confinedto the groups with higherchromosome rov, 1974; Goldblatt,1981, 1984, 1985, 1988; numbers.In groupswith lower chromosome num- Goldblatt& Johnson.1990, 1991). These represent bers (n = 4, 9, and 10), we have notfound any B over20% of the species and 53% of the generain chromosomes,although we have examine the the tribe.Excluding ambiguous and erraticcounts chromosomesof morethan 2000 individuals(Wa- 588 Annals of the MissouriBotanical Garden

101 I I I I I I I I I * 1 a I 0 N N

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FIGURES 10-20. Haploid karyotypicidiograms.-10. Podachaeniumeminens (n 19).- 1. Neomirandeabiflora (n = 25).-12. Neomirandeaguevarii (n = 25). 13. Neomirandeaangularis (n 25).-14. Ageratinascorodontoides (n = 17).-i15. Neomirandeaparasitica (n = 17). 16. Ageratinahavanensis (n 17). 17. Mikania congesta(n = 17).-18. Bartlettinasordida (n =16).-19. Stevia salicifolia(nI 12). 20. Steviapilosa (n =11). Scale bar= 4 jim. Volume 82, Number4 Watanabe et al. 589 1995 ChromosomalCytology in Eupatorieae

21111111 2lhhhhhhlhl

23111 1 24II h hhhEU

251 1 1 5 * 1* * * 261I E E *E *I E

271 . u *mu- 28- a - - * *

29- a 9m - - - - ' 301]

FIGURES 21-30. Haploid karyotypicidiograms. 21. Carphochaetebigeloviii (nt 11). 22. Aistroeupatoriumin- ulaefolium(n = 10). 23. Acanthostylesbuniifolius (n = 10). 24. Eupatoriumpurplureum (n = 10). 25. Adenostemma cuatrecasasii(n = 10). 26. Hebecliniummacrophyllum (n, = 10). 27. Koanophyllonlongifolium (r = 10). 28. Lepidesmiasquarrosa (n = 10). 9. Fleischmanniasideritides (n, = 10). 30. Brickelliachloroleppis (nr = 9). Scale bar = 4 jim.

tanabe, 1986; Watanabe et al. 1990; the present been reveale(1within this genus. The subdivision and unpublished data). of Neomirandeabased on chromosomenumber is In the genus Neomirandea, all of the species ex- supportedby many synapomorphiemutations in amined have a variable number of B chromosomes chloroplastDNA (Yaharaet al., in prep.). between individuals. It is uncertain at present what Dibase chromosomenumbers of 4 and 10, and role B chromosomes play in chromosomal evolution of 11 and 12, are also confirmedin Fleischmannia in the groups with higher chromosome numbers. and Stevia.Although there are manyreports of 2n To obtain an accurate picture of the nature of = 34, suggestingthe occurrenceof x = 17 in Stev- higher base numbers in Eupatorieae, we have stud- ia, thosereports appear to be miscounts. ied the genus Neomirandea more intensely.In pre- In the genusAgeratina, we examinedeight spe- vious studies, a wide range of chromosome num- cies. Ageratinarothrockii has twocytotypes 3x and bers, fromn = 14 to 26, has been reported,arid it 4x based on x = 17. Ageratumcorymbosum (2n = has been uncertain what the true base chromosome 4x = 40), Chromolaenalaevigata (2n 5x = 50), number is forthis genus. The six species examined Koanophyllonlongifolium (2n = 3x 30), and have a chromosome number of either 2n = 34 or Praxelisclematidea (2n = 3x = 30) are polyploids 50. Previously reported chromosome numbers are based on x = 10. also around n = 17 or n = 25, except for n = 14 The distributionof base chromosomenumbers in in N. ovadensis R. M. King & H. Rob. (Strother, the species, genera,and subtribesis shownin Fig- 1983) and n = 20 in N. burgeriR. M. King & H. ure 1. Amongthem, the base numbersof x = 8, Rob. (King et al., 1976). Thus, two differentlin- 19, and 20 reportedin a few species and genera eages based on dibase chromosome numbers have appear to be miscounts.Although there is a small 590 Annals of the MissouriBotanical Garden

Table 2. Karyotypedata forthe helianthoidspecies Podachaeniumeminens and 20 representativeeupatorioid species. *: B chromosomesare excludedfrom these measurements. **: Because Koanophyllonlongifolium is regarded as an autotriploid,its totalkaryotypic length is calculatedas two-thirdsof totalgenome.

Chromo- Mean arm some Totalkaryotypic Range ratio number length(pum) longest-shortest long arms/ Species (2n) mean + S.E. (ratio) shortarms Podachaeniumeminens 38 96.22 ? 2.25 3.21-1.91 (1.68) 1.47 Neomirandeabiflora* 50 107.06 + 4.02 3.30-1.40 (2.36) 1.51 N. guevarii* 50 103.60 + 2.93 3.24-1.20 (2.70) 1.48 N. angularis* 50 102.22 + 3.23 2.73-1.62 (1.69) 1.52 N. parasitica* 34 83.84 + 1.87 3.30-1.81 (1.82) 1.71 Ageratinascorodonioides 34 88.20 + 2.38 3.57-1.96 (1.82) 1.90 A. havanensis 34 83.48 1 2.20 3.37-1.87 (1.80) 1.38 Mikania congesta* 34 52.62 ? 1.67 2.40-1.14 (2.11) 1.31 Bartlettinasordida* 32 97.62 ? 2.48 4.51-2.05 (2.20) 1.41 Steviasalicifolia 24 53.24 ? 1.63 2.82-1.60 (1.76) 1.52 S. pilosa 22 42.50 1 0.60 2.87-1.43 (2.01) 1.40 Carphochaetebigelovii 22 117.14 1 4.01 7.12-4.43 (1.57) 1.70 Austroeupatoriuminulaefolium 20 77.14 1 4.13 4.89-3.27 (1.50) 1.79 Acanthostylesbuniifolius 20 76.98 ? 2.61 4.83-2.96 (1.63) 1.56 Eupatoriumpurpureum 20 63.78 1 3.06 3.98-2.53 (1.57) 1.74 Adenostemmacuatrecasasii 20 47.78 1 0.76 2.82-1.92 (1.47) 1.34 Hebecliniummacrophyllum 20 42.60 1 2.15 2.54-1.64 (1.55) 1.63 Lepidesmiasquarrosa 20 30.98 ? 1.63 1.68-1.16 (1.45) 1.32 Koanophyllonlongifolium** 30 29.46 ? 0.91 1.72-1.14 (1.51) 1.49 Fleischmanniasideritides 20 27.68 ? 0.73 1.59-1.17 (1.36) 1.42 Brickelliachlorolepis 18 42.66 + 1.33 2.87-2.07 (1.39) 1.31

peak at x = 17, 12 ofthe 18 subtribes,73% ofthe tribeby most previousworkers. In addition,this reportedgenera, and 53% of the reportedspecies numberhas been proposedas the base numberfor have chromosomenumbers based on x = 10. Thus, theentire (Robinson et al., 1981). High the predominantchromosome number among spe- chromosomenumbers, such as n = 15, 16, 17, and cies, genera,and subtribes,x = 10, was doubt- 25, have been consideredto be the resultof poly- lesslyregarded as the ultimatebase numberin the ploid increase followedby dysploidloss (King &

Table 3. Distributionof base chromosomenumbers in Eupatorieaeaccording to subtribe(King & Robinson,1987). Chromosomenumbers underlined are confirmedby the presentstudy.

Adenostemmatinae 10 Eupatoriinae 10 Disynaphiinae 10 Gyptidinae 10 Ageratinae 9 10 11 12 Ayapaninae 10 Alomiinae 9 10 Liatrinae - 10 Fleischmanniinae 4 10 Critoninae 10 Praxeiinae 10 Hebeclinae 10 16 Trichoroninae 15 Neomirandeinae 17 25 Mikaniinae 17 18 Oxylobinae 16 17 Hofmeisteriinae 18 Oaxacaniinae 18 Volume 82, Number4 Watanabe et al. 591 1995 ChromosomalCytology in Eupatorieae

Robinson,1987). Taxa withhigh chromosome num- n = 10, the chromosome length of species shows bers thatare classifiedas differentsubtribes are, wide successive ranges from4.89 to 1.14 pm, al- however,distinct morphologically from the taxa though there is a significantlength differencebe- withn = 10. This is in contrastto the present tween the species with longer and shorter chro- polyploidderivatives based on x = 10, 11, and 17, mosomes. which are rathersimilar morphologically to their To assess relationships between the different diploidancestors. On theother hand, some workers chromosome numbers and lengths, we have mea- (Grant,1953; Turner& King,1964) have postulat- sured the total karyotypiclength of these species. ed thatspecies withthe ancestralbase numberof Total karyotypiclength of Podachaenium eminens x = 5 and 4 mighthave givenrise throughsuc- (2n = 38, Heliantheae) is comparable to that of cessive allopolyploidyto thosetaxa withbase num- Ageratina scorodontoideswith 2n = 34, as well as bers of 9, 10, and 17. It is difficultto imagine, that of Bartlettina sordida with 2n = 32. In addi- however,that the Costa Rican species of Fleisch- tion, total karyotypiclength of those species with manniahaving n = 4 are primitivebecause oftheir higher chromosome numbers is also comparable to specializedannual habitand weediness. that of Austroeupatoriuminulaefolium and Acan- In the course of electrophoreticstudies on Eu- thostylesbuniifolius with 2n = 20. It appears that patorium,we foundthat diploid species of Eupa- the differencein chromosome number and length toriumwith a chromosomenumber of n = 10 had between the taxa is due to descending dysploidy extensivegene duplications(Yahara et al., 1989). caused by very unequal reciprocal translocation This findingcasts furtherdoubt on the hypothesis with loss of minute centromeric fragments. This thatx = 10 was the ultimatebase numberfor Eu- contrasts with the previous hypothesis (King & patorium,as well as Eupatorieae,because diploid Robinson, 1987) that the higher chromosome num- vascularplants have a minimumhighly conserved bers of n = 16 to 18 have been derived fromn = numberof isozymes(Gottlieb, 1981). 10 by polyploidization followed by dysploid loss. The chloroplastDNA data providesufficient res- The comparison between the karyotypeswith 2n = olution to determinethe ancestral chromosome 24 and 22 in Stevia tends to substantiate partlythis numberand the directionalityof dysploidywithin conclusion. The shrubby Stevia salicifolia with 2n Eupatorieae(Ito et al., in prep.).Specifically, in the = 24 has a unimodal karyotype,and the herba- cpDNA phylogeny,those taxa withn = 17 in Eu- ceous S. pilosa with 2n = 22 has a bimodal karyo- patorieaeoccur at thebase ofthe tribe next to three type with two longer pairs of chromosomes. These helianthoids,and taxa withlower numbers, such as longer chromosomes in the species with n = 11 n = 10, are derivedgroups. Therefore, x = 17 is may be the result of translocations to their long acceptedas the ultimatebase numberin the tribe, arms fromeach arm of one chromosome of the spe- and chromosomenumber evolution has proceeded cies with n = 12. Sclerolepis unitfora(2n = 30) has in a descendingdirection only in thistribe, except also a bimodal karyotypewith one longer pair of forn = 25 in Neomirandea.The highestbase num- chromosomes, suggesting a translocated product. ber of x = 25 in Neomirandeacould be derived Throughoutthe process of evolutionaryadvance- fromx = 17 by ascendingdysploidy as suggested ment a reduction in chromosome size has also oc- fromthe higher degree of gene duplications (Suzuki curred in a similar manner in several genera, such et al., unpublished)and the significantlylonger to- as Mikania, Polyanthina,Hebeclinium, Fleisch- tal karyotypiclength in species withn = 25 as mannia,Ageratum, and Conoclinium.With the ge- comparedto species withn = 17. nus Brickellia, Gaiser (1953, 1954) noted the cor- Differencesin chromosomesize and shape have relation between the trend toward reduction in been mentionedin the studies of Eupatorieaeby chromosome size and change of habit fromshrubby Grant(1953) and Gaiser (1953, 1954). Grantre- to herbaceous. Annual species have shorter chro- portedtwo size rangesfor chromosomes. For spe- mosomes. This is true in the genus Stevia, too (Ta- cies in Eupatoriumwith n = 10, the size ranged ble 2, in the comparison between S. salicifolia with from2.5 to 6.5 pm. For species in Ageratinawith 2n = 24 and S. pilosa with 2n = 22, t = 5.514; p n = 17, the size rangedfrom 1.5 to 3.1 pm. For < 0.01). The understoryshrub Carphochaete bige- species of Conocliniumwith n = 10, the size lovii has the longest chromosomal and total karyo- rangedfrom 1.0 to 2.0 pm. We also confirmedthe typic length among the examined taxa. In Eupato- significantchromosome difference between genera rieae, chromosomal evolution has followed a (Table 2). Amongthe species with n = 17, the general trend of reduction in chromosome number chromosomelength of Mikania congestais signifi- and length. cantlyshorter than others. Among the species with Of course, polyploidy has occurred frequentlyin 592 Annals of the MissouriBotanical Garden

several lineages as shown in Table 1. Polyploidy bers. 1984-1985. Monogr.Syst. Bot. Missouri Bot. Gard. 23. accompanied by agamospermy is common in the & D. Johnson,Editors. 1990. Index to Plant genera Ageratina, Stevia, Chromolaena, Praxelis ChromosomeNumbers, 1986-1987. Monogr.Syst. Bot. (King & Robinson, 1987), and Eupatorium (Wata- MissouriBot. Gard. 30. nabe, 1986). The frequent occurrences of poly- & , Editors. 1991. Index to P'lantChro- mosomeNumbers, 1988-1989. Monogr.Syst. Bot. Mis- ploids including odd-ploids in these genera are souriBot. Card. 40. consistent with their previous inferences of wide- Gottlieb,L. D. 1981. Electrophoreticevidence and plant spread agamospermy. populations.Progr. Phytochem. 7: 1-46. The shrubby or arborescent habit is character- Grant,W. F. 1953. A cytotaxonomicstudy in the genus istic of taxa with high chromosome numbers in rel- Eupatorium.Amer. J. Bot. 40: 729-742. Jansen,R. K., H. J. Michaels& J. D. Palmer.1991. Phy- atively stable habitats both in Heliantheae and Eu- logenyand characterevolution in the Asteraceaebased patorieae. Primitive groups of Eupatorieae follow on chloroplastDNA restrictionsite mapping.Syst. Bot. similar evolutionaryattributes to some members of 16: 98-115. Heliantheae. The reduction in chromosome number Karis, P. 0. 1993. Morphologicalphylogenetics of the Asteraceae-Asteroideae,with notes on characterevo- correlated withthe herbaceous habit in Eupatorieae lution.P1. Syst.Evol. 186: 69-93. might promote the exploitation of open habitats Kim, K. J., R. K. Jansen,R. S. Wallace, H. J. Michaels with ephemeral resources. & J. D. Palmer. 1992. Phylogeneticimplications of Cytology might prove most helpful in subtribal rbcl,sequence variationin the Asteraceae.Ann. Mis- souriBot. Card. 79: 428-445. and generic circumscription,and in reflectingsome King, R. M. & H. Robinson. 1987. The generaof Eu- of the evolutionarytrends. patorieae(Asteraceae). Monogr. Syst. Bot. MissouriBot. Gard. 22: 1-581. D. W. Kyhos,A. M. Powell,P. H. Raven & H. LiteratureCited Robinson.1976. Chromosomenumbers in Compositae. XIII. Eupatorieae.Ann. MissouriBot. Gard. 63: 862- Bremer,K. 1987. Tribalinterrelationships of the Aster- 888. 3: 210-253. aceae. Cladistics Robinson,H., A. M. Powell,R. M. King & J. F. Weedin. Cladisticsand Classification. . 1994. Asteraceae. 1981. Chromosomenumbers in Compositae,XII: He- TimberPress, Portland, Oregon. liantheae.Sniithsonian Contrib. Bot. 52: 1-28. , R. K. Jansen,P. 0. Karis, M. Kallersjo,S. C. Strother,J. 1.. 1983. Morechromosome studies in Com- & Keeley,K. J. Kim, H. J. Michaels,J. D. Palmer R. positae.Amer. J. Bot. 70: 1217-1224. S. Wallace. 1992. A reviewof the phylogenyand clas- Turner,B. L. & R. M. King. 1964. Chromosomenumbers sificationof the Asteraceae. Nordic J. Bot. 12: 141-148. in theCompositae. VIII. Mexicanand CentralAmerican Fedorov,A. A., Editor. 1974. ChromosomeNumbers of species. Southw.Naturalist 9: 27-39. FloweringPlants. Otto Koeltz Scientific Publ., Koenig- Watanabe,K. 1986. Cytogeographyof the genus Eupa- stein. toritrn(Compositae). A review.P1. Sp. Biol. 1: 99-116. Gaiser,L. 0. 1953. Chromosomestudies in Kuhniinae C. R. Carter& S. Smith-White.1975. The cy- (Eupatorieae).I. Brickellia.Rhodora 55. 253-267, 269- tologyof Brachycomelineariloba. 5. Chromosomerela- 288, 297-321, 325-345. tionshipsand phylogenyof the race A cytodemes(= . 1954. Studies in the Kuhniinae(Eupatorieae). 2). Chromosoma(Berl.) 52: 383-397. II. J. ArnoldArbor. 35. 87-133. , M. Ito,T. Yahara,V. Sullivan,T. Kawahara& D. Goldblatt,P., Editor. 1981. Index to Plant Chromosome J. Crawford.1990. Numericalanalyses of karyotypeof Numbers,1975-1978. Monogr.Syst. Bot. MissouriBot. the genus Eupatorium(Compositae, Eupatorieae). PI. Gard. 5. Syst.Evol. 170: 215-228. Editor.1984. Indexto PlantChromosome Num- Watson,I. E., R. K. Jansen& J. R. Estes. 1991. Tribal bers, 1979-1981. Monogr.Syst. Bot. Missouri Bot. placementof Marshallia (Asteraceae) using bhloroplast Gard.8. DNA restrictionsite mapping.Amer. J. Bot. 78: 1028- Editor.1985. Indexto PlantChromosome Num- 1035. bers, 1982-1983. Monogr.Syst. Bot. Missouri Bot. Yahara,T., T. Kawahara,D. J. Crawford,M. Ito & K. Wa- Gard. 13. tanabe. 1989. Extensivegene duplicationsin diploid Editor.1988. Indexto PlantChromosome Num- Eupatorium(Asteraceae). Amer. J. Bot. 76: 1247-1253.