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タイトル Very Low Genetic Heterozygosities in Sexual and Agamospermous Title Populations of altissimum () 著者 Yahara, Tetsukazu / Ito, Motomi / Watanabe, Kuniaki / Crawford, Daniel Author(s) J. 掲載誌・巻号・ページ American journal of botany,78(5):706-710 Citation 刊行日 1991-05 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 JaLCDOI URL http://www.lib.kobe-u.ac.jp/handle_kernel/90001249

PDF issue: 2021-09-25 AmericanJournal of Botany 78(5): 706-7 10. 199 1.

VERY LOW GENETIC HETEROZYGOSITIES IN SEXUAL AND AGAMOSPERMOUS POPULATIONS OF EUPATORIUM ALTISSIMUM (ASTERACEAE)1

TETSUKAZU YAHARA,2 MOTOMI ITO, KuNIAKI WATANABE, AND DANIEL J. CRAWFORD BotanicalGardens, University of Tokyo, 1842 Hanaishi-cho,Nikko 321-14, Japan; MakinoHerbarium, Tokyo Metropolitan University, Setagaya, Tokyo 158, Japan; BiologicalInstitute, Faculty of General Education, Kobe University,Kobe 657, Japan;and Departmentof Botany, The Ohio StateUniversity, Columbus, Ohio 43210-1293

An electrophoreticstudy revealed very low geneticheterozygosities for both sexualand agamospermouspopulations of Eupatorium altissimum in easternNorth America. Low het- erozygosityorgene diversity (HT = 0.03) canbe explainedby the small sizes of the extant sexual populations,estimated to be on theorder of 104. Therewas no substantialdifference in observed heterozygositybetween sexual and agamospermousraces. This result shows that a higherlevel ofheterozygosity is not a prerequisitefor the evolution of agamospermous races from the sexual ancestor.

Apomixisby seed (agamospermy)or spore higherheterozygosity than their sexual ances- (agamospory)has been documentedfor nu- tors. meroustaxa belongingto variousgroups of It has beentheoretically postulated that ap- angiospermsand pteridophytes.Several am- omicticmutants can easilyinvade sexual pop- phi-agamiccomplexes were the subjects of ex- ulationsbecause they do notproduce males or tensivestudies until the 1950s due to their thereis twofoldparent-offspring relatedness (see taxonomicaland cytologicalcomplexities. Williams,1975; Maynard Smith, 1978, 1984). These classicalworks are reviewedby Gus- Fromthis theoretical perspective, higher het- tafsson(1946-1947), Stebbins (1950), Nygren erozygosityis not a prerequisitefor the evo- (1954),and morerecently by Grant (1981) and lutionof apomictic races from a sexualances- Nogler(1984). These authorsconcluded that tor.Thus it is ofparticular interest to compare thoseapomictic taxa examined genetically are heterozygositiesofsexual and agamospermous all highlyheterozygous. Recent electrophoretic races within a singlespecies. studiesof some amphi-agamicplant groups Amongthe 23 speciesof NorthAmerican also reportedthat agamospermous taxa have EupatoriumL., both sexual and agamosper- higherheterozygosities than their sexual rela- mousraces have been reported for nine species. tives(Bayer and Crawford,1986; Watano and In thesespecies, sexual and agamospermous Iwatsuki,1988; Bayer, 1989a; see also Vrijen- racesare indistinguishablein grossmorphol- hoek,1990 for evidence from parthenogenetic ogy. These species provide a goodopportunity animals). These agamospermousraces are, to examineheterozygosities ofsexual and aga- however,probably allopolyploids that main- mospermouspopulations within a singlespe- taina highlevel of fixedheterozygosity even cies.As thefirst attempt to compareheterozy- in thesexual state (Roose and Gottlieb,1976). gositiesbetween sexual and agamospermous Therehave beenlimited data comparinghet- populationsof these nine species, we examined erozygositiesof sexual and agamospermous allozyme variation in Eupatorium altissi- races withina singlespecies (Bayer,1989b; mumL. Vrijenhoek,1990), and it is still uncertain whetheragamospermous races in generalhave MATERIALS AND METHODS Sexual Eupatoriumaltissimum is an out- I Receivedfor publication 2 April 1990; revisionac- crossing(self-incompatible) perennial without cepted15 January1991. vegetativereproduction. This species occurs as The authorsthank R. J.Bayer and B. J.Ray forhelpful sexualdiploid with normal pollen and commentson earlier versions ofthe manuscript. This study was supportedby grants from the Ministry of Education, triploidplants that have no pollen,or less fre- Cultureand ScientificResearch, Japan (No. 63041048) quently,malformed abortive pollen grains; the and byNSF grantBSR-8521 152. latterproduce fertile seeds and thus are re- 2 Authorfor correspondence. gardedas agamospermous(Sullivan, 1976).

706 May 1991] YAHARA ET AL. -HETEROZYGOSITY IN EUPATORIUM ALTISSIMUM 707

TABLE 1. Collectioncodes and localities of sampled pop- thethree buffer systems described by Yahara ulations of Eupatorium altissimum et al. (1989). SystemI resolvedphosphoglu- coisomerase(PGI) and triosephosphateisom- Collec- tion erase(TPI), systemII resolvedalcohol dehy- codes Localities drogenase(ADH) and phosphoglucomutase Sexual populations (PGM), and systemIII was employedto re- 45 US 65S, 10 miles S of JCT 60, ChristianCo., solve aconitase(ACN), isocitratedehydroge- MO nase (IDH), and shikimatedehydrogenase 46 US 65S, 13 miles S of JCT 60, ChristianCo., (SKDH). Stainingschedules followed Soltis et MO 47 Rt. 248, 3 miles N of JCT 65, Taney Co., MO al. (1983). 48 US 65S, 8 miles N of Bear Creek Spring,near Examinationof gels stained for ACN, IDH, Burlington,Boone Co., AR PGI, PGM, and TPI providedreliable genetic 49 US 62E, 8 miles W of Yellville, Marion Co., datafor 13 isozymeloci. Many diploid species AR of NorthAmerican Eupatorium show exten- 50 US 62E, 3 miles W of Yellville, Marion Co., siveduplications for genes encoding IDH, PGI, AR and PGM (Yahara et al., 1989). In sexualE. 51 Rt. 5S, 3.8 miles S of Mountain Home, Bax- ter Co., AR altissimum,three isozymes were detected for 53 Rt. 5S, 2.7 miles S of White River, Stone Co., theseenzyme species. Based on these data, Nei's AR statisticsof geneticidentity (Nei, 1972) and Agamospermouspopulations genediversity (Nei, 1973) werecalculated for 55 Rt. 14S, 17 miles NW of Batesville, Stone sexualdiploid populations, and meanhetero- Co., AR zygosities(mean of observedheterozygosity 57 IS 55N, 1 mile S of St. Mary, Ste. Genevieve overall lociexamined) were calculated for sex- Co., MO ual andagamospermous populations. Gene di- 58 IS 70E, 1 mile W of Altamont,Effingram Co., versitywas calculated using all 13 locifor com- IL parisonwith theoretical expectations, and using 59 IS 70E, 2 miles E of JCT 231, Putnam Co., IN polymorphicloci to comparewith the data re- OH Campus of Ohio State University,Columbus, viewedby Hamrick and Godt(1990). The en- OH zymesADH and SKDH werenot interpreted geneticallydue to overlappingexpression of differentloci, and thuswere not includedin thecalculation. These agamospermousplants occur widely in easternNorth America, but sexual diploids are RESULTS restrictedto the Ozark Mountainslying in southernMissouri and northern Arkansas, and Of the 13 loci scored,six were polymorphic to a smalldisjunct area in centralTennessee. in sexual populations(Table 2). At each of We sampled259 individualsfrom eight pop- thesesix loci, a commonallele occurs in a mean ulationsof the Ozark Mountains. Examination frequencyhigher than 0.90 (Table 2). Mean ofpollen fertility indicated that all samplesare and rangesof geneticidentities for pair-wise sexual, and chromosomecounts confirmed comparisonsof populations and gene diversity themas diploids(2n = 20). These samples (expectedheterozygosity) statistics for sexual covera largepart of the rangeof diploidE. populationsofE. altissimumare given in Table altissimumin theOzarks. We also sampled 144 3. The data show thateight populations are individualsfrom five agamospermous popu- geneticallyvery homogeneous;there is no lations(Table 1; see Table 4 forsample size of markeddifferentiation among populations, and each population).Chromosome counts con- gene diversity(heterozygosity) is very low. firmedthem as triploids(2n = 30), and they MeanHTfor previously published data for pre- lackedpollen. dominantlyoutcrossing, animal-pollinated Plantsbearing flower buds and young,green speciesis 0.310 (Hamrickand Godt, 1990), leaves werecollected in thefield, transported and is 4.6 timeshigher than in E. altissimum. to the laboratoryon ice, and storedin a re- For agamospermousplants, four genotypes frigeratoruntil used forelectrophoretic anal- wererecognized using 13 enzymeloci, and all ysis. For electrophoresis,flower buds and a populationswere monoclonal (Table 2). Plants smallpiece of leaf material of individual plants fromIndiana and Ohio wereidentical. Three wereground in 1.0 mlof cold extraction buffer genotypesfrom the agamospermousplants as describedby Odryzykoskiand Gottlieb (otherthan the Illinois type) exhibited one or (1984) and modifiedby Yahara et al. (1989). tworare alleles from the sexual populations in Enzymeswere resolved in 12%starch gels using the heterozygouscondition. Mean observed 708 AMERICAN JOURNAL OF BOTANY [Vol. 78

TABLE 2. Mean allele frequenciesat 13 enzyme loci in sexual E. altissimum and correspondingelectrophoretic phenotypesin agamospermousE. altissimum.Mean allelefrequencies for eight populations are givenin parentheses

Alleles found in agamospermouspopulation Locus Allele frequenciesin sexual population AR MO IL IN OH Aco-l a (0.048),b (0.952) b b b b b Aco-2 a(0.993), b (0.007) a a a a a Idh-l a (1.000) a a a a a Idh-2 a (1.000) a a a a a Idh-3 a (0.996),b (0.004) a a a a a Pgi-l a (1.000) a a a a a Pgi-2 a (1.000) a a a a a Pgi-3 a(0.993), b (0.007) a a a a/b a/b Pgm-l a (1.000) a a a a a Pgm-2 a (1.000) a a a a a Pgm-3 a(0.946), b (0.054) a a/b a a a Tpi-l a (0.052),b (0.907),c (0.041) b/c a/b b b b Tpi-2 a (1.000) a a a a a Frequencyof corresponding genotypein diploid 0.07 0.02 0.52 0.01 0.01 heterozygosityofagamospermous populations productionbecause it does notproduce males rangedfrom 0.00 to 0.15 witha meanof 0.08 and thereis enhancedparent-offspring relat- (Table 4). Mean observedheterozygosities cal- edness(Williams, 1975; Maynard Smith, 1978, culatedfor eight sexual populations ranged from 1984). Thereis evidencesupporting this the- 0.01 to 0.14 witha mean of 0.07, and the oreticaladvantage of agamospermousplants differencefrom agamospermous populations (Michaels and Bazzaz, 1986; Bierzychudek, was not significantby Mann-WhitneyU test. 1989, 1990; Yahara, 1990). In E. altissimum, This statisticalresult and completehomozy- therewas no substantialdifference in hetero- gosityin theIllinois type indicates that there zygositybetween sexual and agamospermous is no substantialdifference in heterozygosityraces. This resultclearly shows that a higher betweensexual and agamospermouspopula- levelof heterozygosity is not a prerequisiteto tions of E. altissimum. theevolution of agamospermousraces and is consistentwith the above theoreticalperspec- DISCUSSION tive. Agamospermousplants have been consid- TABLE 4. Observedheterozygosities in sexual popula- eredto be advantageousbecause they maintain tionsand observed heterozygosities inagamospermous hybridityor highheterozygosity without the populations of Eupatorium altissimum breakingup of advantageousgene combina- tions by recombination.From a theoretical Observed perspective,however, asexual reproduction it- Populations Sample size heterozygositya selfhas a twofoldadvantage over sexual re- Sexual 45 18 0.07 46 16 0.06 TABLE 3. Geneticidentities and genediversity statistics 47 81 0.07 forsexual populations of Eupatorium altissimum 48 14 0.06 49 10 0.03 Mean geneticidentities for all pair- 0.998 50 66 0.07 wisecomparisons of populations 51 47 0.02 Rangeof genetic identities for all pop- 0.994-1.000 53 10 0.14 ulations Mean forsexual populations 0.07 Totalgene diversity (HT) 0.03la 0.067b Agamospermous Population-levelgene diversity (HS) 0.030a 55 (AR) 24 0.08 0.065b 57 (MO) 24 0.15 Gene diversitybetween populations 0.001a 58 (IL) 24 0.00 (DST) 59 (IN) 24 0.08 Differentiationamong populations 0.049a OH 48 0.08 (GST) Mean foragamospermous populations 0.08 a Calculatedusing 13 loci. bUsing six polymorphicloci. a Mean for 13 loci. May 19911 YAHARA ET AL. -HETEROZYGOSITY IN EUPATORIUM ALTISSIMUM 709

At all 13 loci examined,all fouragamosper- populationsof E. altissimumwere more highly mous raceshad thesame allele thatwas pre- heterozygousat thetime of originof theaga- dominantin the sexual populations. Four other mospermousraces. Available evidence is ad- alleles foundin agamospermousraces were vantageousto the latterhypothesis as stated foundin low frequencies in sexualpopulations. below. This resultand theequivalent level of hetero- Because diploidE. altissimumis a self-in- zygosityin sexual and agamospermouspop- compatible,predominantly outcrossing spe- ulationssuggest nonhybrid origins of the aga- cies,the low level of genetic heterozygosity can mospermousraces. Morphologicalsimilarity be attributedto reducedpopulation size in the betweenthe sexual and agamospermousraces extantpopulation and/or a populationbottle- supportsthis view. Recently,Gastony and neckin thepast (Nei, Maruyama,and Chak- Gottlieb(1985) and Gastony(1988) suggested raborty,1975). Underthe neutral theory, ex- electrophoreticallythat agamospermous races pectedheterozygosity (H) in equilibriumwith ofa fewfern species are ofautopolyploid origin. mutationand geneticdrift is: repro- Althoughassociation between asexual H = 4NeV/(4NeV+ 1), ductionand hybridityhas been documented in plants(see Richards,1986 for a mostrecent whereNe is effectivepopulation size and v is review) and also in many parthenogeneticmutation rate. This equation gives an estimate animals(White, 1978; Vrijenhoek,1990), our ofNeV using estimated expected heterozygosity resultshows that this is not the rule. This or total gene diversity(HT) as: associationcan be secondarybecause hybrid- - izationbetween sexual and apomicticplants (Nev)est= HT/4(1 HT). will produceapomictic hybrids. This process BecauseHT is 0.031 in sexualE. altissimum, is recentlydocumented in severalfern genera NeVis estimatedto be 8 x 10-3. This givesan (Gastonyand Gottlieb,1985; Gastony,1988; estimateof Ne as 6 x 104 assumingv = 1.3 x Watanoand Iwatsuki,1988; Suzukiand Iwat- 10-7 (foran estimateof electrophoretically de- suki,1990) and in angiospermsincluding Eu- tectableneutral mutation rate see Kimuraand patorium(Yahara, 1990). Ohta,1971; Kimura, 1983; Nei, 1987).Sexual It is consideredthat apomicts are likelyto populationsof E. altissimumoccur very spo- becomemore heterozygous than their sexual radicallyaround the Missouri-Arkansasbor- progenitorsover time, because apomicts should der.We observeda fewlarge populations with accumulatemutations in the absence of re- morethan 100 individuals,and thenumber of combination(see Lokki,1976a, b; Vrijenhoek, theselarge sexual populationsin the Ozark 1990 and referencestherein). In thisprocess, Plateauis also estimatedto be severalhundred. apomictsare expectedto gain unique alleles Thusthe estimate of the actual population size whichare not found in their sexual progenitors. (i.e., total number of reproductive individuals) In E. altissimum,however, all alleles of the is on the orderof 10,000,which agrees ap- agamospermousraces are also foundin sexual proximatelywith the estimate of effective pop- populations.This factsuggests that the aga- ulationsize. Thus the reduced genetic diversity mospermousraces are of relativelyrecent or- in sexualE. altissimumcan be explainedwith- igin. out assuminga populationbottleneck in the Anotherinteresting finding in thisstudy is past. thevery low genetic heterozygosity in the sex- Amongthe four agamospermous races stud- ual populationsof E. altissimum.It has been ied threehad alleles foundonly in low fre- consideredthat several recessive genes should quenciesin the sexualpopulations. Expected be broughttogether for the originof agamo- frequenciesof the genotypes corresponding to spermy,and thusthe sexual ancestors of aga- theseagamospermous races are quite low (0.07- mospermsare likelyto be highlyoutcrossing 0.01) inthe extant sexual population (see Table (Powers, 1945; Asker, 1980; Marshall and 2), andit is doubtfulthat these originated from Brown,1981). In thishypothesis, the recessive the extantsexual population.This factmay genes for agamospermyare assumed to be suggestthat the sexual race ranged more widely maintainedin lowfrequencies in ancestralsex- and was moreheterozygous at thetime of or- ual populations,and thusthe sexual ancestors igin of theseagamospermous races. Because are expectedto be highlyheterozygous. The the agamospermousrace in higherplants is resultof this study, however, revealed very low advantageousnot only in enhancedparent-off- heterozygosityin the sexual populations of E. springrelatedness, but also in ensuredfertility altissimum.This resultmay suggest that apo- (Bierzychudek,1987; Yahara, 1990),the sex- mictscan arise fromsexual stockswith very ual racecould survive only in extremehabitats low heterozygosity.Alternatively, the sexual withina small area afterthe originand sub- 710 AMERICAN JOURNAL OF BOTANY [Vol. 78

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