Heredity 77 1996 388395Received 14 November 1995Low genetic variati - PDF document

1 Heredity 77 (1996) 388—395Received 14 No
Heredity 77 (1996) 388—395Received 14 November 1995Low genetic variation in Amentotaxusformosana Li revealed by isozyme analysisand random amplified polymorphic DNAmarkersCHIEH-TING WANGt, WEI-YOUNG WANG1, CHIA-HUA CHIANG, YA-NAN WANG& TSAN-PIAO LIN*trSilv/culture Division, Taiwan Forestry Research Institute, 53 Nan-Hai Road, Taipei and Department of Forestry,National Ta/wan University, Taipei, TaiwanTheobjective of this research was to use random amplified polymorphic DNA (RAPD) andisozyme analysis to investigate genetic variation in narrowly distributed populations of Amento-taxus formosana Li. A total of 20 loci from 10 enzyme systems were analysed in 50 individualtrees from each of the two natural populations. No isozyme variation was observed in theTsatsayalai population. Phosphoglucose isomerase (Pgi-1) was the only polymorphic enzyme inthe Tawu population, giving 5 per cent polymorphic loci with 0.008 expected heterozygosity.No genetic distance was found between these two populations using isozymes. Amentotaxusformosana demonstrated a high proportion of monomorphic RAPD fragments, about 79 percent, for 20 arbitrary oligonucleotide primers. High similarity (0.994) was found between theTawu and Tsatsayalai populations. RAPD markers provided further confirmation of the lowlevels of genetic variation in A. forinosana detected by isozyme analysis. The value of isozymeanalysis was emphasized by the finding of the rare allele, Pgi-la, which was present only in theTawu population. Based on the analysis of 110 individuals, representing 16 per cent of a nativepopulation, it was found that the younger tree category had a higher frequency of Pgi-la(0.125) than the older tree category (0.053), resulting in an expected heterozygosity of 0.250and 0.105, respectively. It was inferred that the appearance of the Pgi-la allele could be theresult of a mutation in the Tawu population and that selection is acting directlyupon treescarrying this allele.Keywords:Amentotaxusformosana, genetic variation, isozyme, RAPD.IntroductionAmentotaxusformosana Li is endemic to Taiwan,where it is probably the most narrowly distributedgymnosperm species. It is highly endangered and hasreceived worldwide attention (Farjon et al., 1993). Itis a dioecious tree producing large and heavy seeds,but is poor in production. It grows in the broad-leaved forests of Taitung Forest District (Tawu) andthe Pingtung Forest District (Tsatsayalai), at anelevation ranging from 900 to 1300 m. The Tawupopulation has about 700 trees greater than 1 cm indiameter and covers an area of about 86 ha. TheTsatsayalai population has about 800 trees and*Correspondence.covers about 225 ha. In 1988 the Council of Agri-culture, Taiwan, designated these two sites asnatural reserves for A. formosana, because the popu-lations were on the brink of annihilation. Theclimate and the components of the plant commu-nities of these two habitats are very similar andbelong to the warm temperate rain forest (Yang,1994). The reversed J-type of the population struc-ture, judged by the frequency distribution of breastheight diameter (DBH) classes, indicated that A.formosana populations can grow continuously andstably under protection in these habitats (Yeh et a!.,1992; Yang, 1994).The primary objective of this research was toinvestigate genetic variation within and betweenthese two populations of A. forinosana, usi

2 ng3881996 The Genetical Society of Great
ng3881996 The Genetical Society of Great Britain. LOW GENETIC VARIATION IN AMEN TO TA XUS FORMOSANA 389isozyme and random amplified polymorphic DNA(RAPD) markers (Williams et al., 1990). Geneticstudies based on isozyme data have major advan-tages over RAPD markers in that they are cheaperand easy to perform, but also give more informationon genotypic relationships. Some evidence hassuggested that allozyme variation may not be able toprovide an accurate or complete measure of nucleo-tide variation in the genome (Wolff, 1991; Heun etal., 1994; Meijer et al., 1994). RAPD markers, on theother hand, may provide a less biased measure ofgenetic variability and a greater resolution of subtlegenetic differences for inferring genetic structure.RAPD analysis has resulted in a more definitivegrouping (Heun et a!., 1994; Maal3 & Klaas, 1995),even though RAPD polymorphism is poorly under-stood and believed to be based upon eithersequence variation or mismatches in the primerbinding sites. However, in this report we presentsome unique information from the isozyme analysisthat it is not possible to obtain using RAPDmarkers.Materials and methodsSamplingThe locations of the two natural populations, Tawuand Tsatsayalai, are shown in Fig. 1. Fifty trees weresampled from each population for isozyme analysis,and 25 and 20 individuals from the Tawu and Tsat-sayalai populations, respectively, for RAPD analysis.Fewer individuals were included in the RAPDs thanthe isozyme analysis because of practical constraints.Random sampling was applied to the trees; however,uniformity was not possible because some trees weregrowing on a steep slope. Young leaf tissue wascollected in April 1994 and stored at —20°C untilrequired and additional seeds were collected inJanuary 1995 for gametophyte analysis. Young leaftissue of an additional 60 individuals in the Tawupopulation was collected in April 1995, and a totalof 110 individuals, which varied from cm DBH, were used to compare the genotypedistribution and allele frequencies of Pgi-l.Isozymeelectrophoresis methodsHorizontalstarch gel electrophoresis was usedto examine ten enzyme systems, namely: esterase(EST, EC 3.1.1.1); fluorescent esterase (F-EST,EC 3.1.1.1); L-aspartate aminotransferase (AAT, EC2.6.1.1); isocitrate dehydrogenase (IDH, EC 1.1.1.42); malate dehydrogenase (MDH, EC 1.1.1.37);The Genetical Society of Great Britain, Heredity, 77, 3 88—395.6-phosphogluconate dehydrogenase (6PGD, EC1.1.1.43); phosphoglucose isomerase (PGI, EC5.3.1.9); phosphoglucomutase (PGM, EC 5.4.2.2);shikimate dehydrogenase (SKDH, EC 1.1.1.25);superoxide dismutase (SOD, EC 1.15.1.1). Youngleaf tissue and megagametophytes were ground withextraction buffer (Feret, 1971). Electrophoresis andstaining followed the procedures described byCheliak & Pitel (1984).Isozymedata analysisAllelefrequencies were calculated for each locusand population. The following four measures wereused to quantify genetic variation within a popula-tion: (1) the expected heterozygosity (Nei, 1975) ateach locus was calculated askHe=1 D2Ii= 1where P, is the frequency of the ith allele, summedover k alleles; (2) the mean number of heterozygousloci per individual was calculated (Nei, 1973); (3)the mean number of alleles per locus was calculatedby averaging over all polymorphic and monomorphicloci; and (4) the effective number of alleles per locus(Ae; Crow & Kimura, 1970), w

3 as defined asAe 1/P?.The number of allel
as defined asAe 1/P?.The number of alleles is maximized when the allelefrequencies at any locus are equal. Both Wright's(1969) F-statistics and Nei's (1978) unbiased geneticidentity (Ia) and genetic distance (D,,) were used toquantify the degree of differentiation among popula-tions. The above calculations, with the exception ofthe effective number of alleles, were performedusing BIOSYS-1 (Swofford & Selander, 1989).DNApreparationTotalcellular DNA was prepared from 0.8 g ofyoung leaf material using a modified mini-CTABmethod (Murray & Thompson, 1980). Leaves werefrozen in liquid nitrogen, ground to a fine powderand suspended in 15 mL extraction buffer (50 mMTris—HC1, pH 8.0, 350 mrvt sorbitol, 5 mivi sodiumEDTA, 10 per cent polyethylene glycol 3350, 0.1 percent bovine serum albumin, 0.1 per cent spermine,0.1 per cent spermidine and 0.1 per cent 2-mercap-toethanol). The extraction was filtered through mira-cloth and centrifuged at 13 000 g for 15 mm in aKontron H-401 centrifuge. The pellet was resuspen-ded in 350 L resuspension buffer (50 mi Tris— 390 C.-T.WANG ETAL.HC1,pH 8.0, 25 mivi EDTA, 350 mrvi sorbitol and 0.1per cent 2-mercaptoethanol) and the nuclei andorganelles lysed by addition of 25 #L 20 per centsarkosyl (N-lauryl sarcosinate) and incubating atroom temperature for 15 mm. After adding 70 L 5M NaC1 and 55 uL 8.6 per cent CTAB (cetyltrime-thylammonium bromide) and heating at 60°C for 10mm, the homogenate was extracted with 600 Lchloroform:isoamyl alcohol (24:1) and centrifuged ina Kubota KM-15200 microcentrifuge at 5000 g for10 mm. The nucleic acid was precipitated from theaqueous phase by adding 400 tL isopropanol andpelleted by centrifugation at 12000 g for 10 mm inthe microcentrifuge, then washed with 70 per centFig. 1 The natural reserves for Amen-totaxus forinosana. A, Tawu; B,Tsatsayalai.absolute ethanol. The pellet was dried and dissolvedin 100 1iL TE buffer (10 mrvi Tris—HC1, pH 8.0, 1mM disodium EDTA), containing 20 mglmL RNase,and stored at —20°C.The DNA concentration wasdetermined using a Hoeffer fluorometer and adjus-ted to 10 ng/jiL for use in the polymerase chainreaction (PCR).Polymerasechain reactionPCRconditions for RAPD reaction with the IdahoAir Thermal Cycler are described as follows. Eachsample, comprising 50 mM Tris—HC1 buffer (pH 8.5)containing 20 mivi KCI, 1.5 mrvt MgCl2, 0.5 mg/mLThe Genetical Society of Great Britain, Heredity, 77, 388—395. N0I23KM LOW GENETIC VARIATION IN AMENTOTAXUS FORMOSA NA 391Table 1 Sequences and codes of random primers and the number ofmonomorphic and polymorphic fragments amplifiedPrimerSequence(5' —3')No. ofmonomorphicfragmentsNo. ofpolymorphicfragmentsTotalOPE-2GGTGCGGGAA9615OPE-12TFATCGCCCC21223OPE-17CTACTGCCGT505OPE-19ACGGCGTATG15318OPS-1CTACTGCGCT12012OPS-lOACCGTr'CCAG10717OPS-13GTCGTTCCTG718OPS-18CTGGCGAACT10313OPY-2CATCGCCGCA13114OPY-7AGAGCCGTCA707OPY—9AGCAGCGCAC14216OPY-lOCAAACGTGGG15015OPY-17GACGTGGTGA19120P-4CGAAGCTTCG13114P-6CCGTCGACGA5813P-bATTGCGTCCA19019P-liATGTCCTCGA10010P-13TCAGCGTGCT909P-14TACCGAACGT81927P-25GGTACCGTGC8614Total229(79.2%)60(20.8%)289BSA, 200 tM each of dATP, dCTP, dGTP, dTTP,0.4 M 10-base primer, 60 ng of template DNA and1.7 units of Taq DNA polymerase (BoeringerMannheim Biochemica) at a final volume of 20 1iL,was heat-sealed in a 25 jiL glass capillary tube.Twenty random primers, 13 (OPE-2, ...,OPY-17)supplied by Operon Technologies and 7 (P

4 -4,P-25) synthesized by Oligos Etc., wer
-4,P-25) synthesized by Oligos Etc., were included inthe survey (Table 1). The amplification conditionsincluded a total of 45 cycles with template denatura-tion at 94°C for 60 s, primer annealing at 37°C for 7s, and primer extension at 72°C for 70 s during thefirst two cycles. The time for template denaturationwas then reduced to 1 s for the remaining 43 cycles.Reactions were further incubated at 72°C for 4 mmand the capillaries were stored at 4°C before theamplification products were analysed by gelelectrophoresis.Analysisof PCR productsPCRproducts were separated using 1.5 per centNuSieve 3:1 agarose (FMC BioProducts) gels byThe Geneticaf Society of Great Britain, Heredity, 77, 388—395.electrophoresis in 1 x TBE buffer, and detected bymeans of ethidium bromide staining, viewed underultraviolet light. Specific amplification products werescored as present (1) or absent (0) in each DNAsample and similarity coefficients (SC)wereestima-ted using Nei & Li's (1979) matching coefficientmethodSC= 2NABI(NA+NB),where NA is the number of bands in individual A,NB is the number of bands in individual B, and NABis the number of bands present in both A and B.Within-population similarity (5)wascalculated asthe mean of SC across all possible comparisonsbetween individuals within a population. Between-population similarity, corrected for within-popula-tion similarity, wasS=1+S'—0.5 (S,+S1),where S, and S1 are the values of S for population iand j,respectively,and S 'isthe average similaritybetween randomly paired individuals from popula-tions i andj (Lynch, 1990). 392 C.-T. WANG ETAL.Table 2 Allele frequencies and the expected (He) andobserved (H0) heterozygosities of the polymorphic locus inthe two populations of Amentotaxus form osanaLocus andallelePopulationAvg.TawuTsatsayalaiPgi-1a0.0900.0000.045b0.9101.0000.955H00.1800.0000.090He0.1640.0000.086Avg. H00.0090.0000.0045Avg. H00.0080.0000.004Expected heterozygosity for each population wascalculated as the arithmetic mean at the 20 loci.ResultsIsozyme analysisIsozyme patterns from gametophyte and leaf tissuewere compared to define the enzyme loci. With theexception of PGI, no differences in band numberwere found between the gametophyte and leaftissue. The number of loci was determined accordingto Weeden & Wendel (1989).Ten enzyme systems, with a total of 20 putativeloci, were stained with consistently good resolution:two loci for PGM, PGI, IDH, AAT, 6PGD andSOD; three for MDH and EST; and one locus forSKDH and F-EST. All loci were monomorphic, withthe exception of Pgi-1, which resolved two cathodallymigrating alleles.The observed allele frequencies, observed andexpected heterozygosities at the polymorphic locus,and average heterozygosities at the population levelare listed in Table 2. Allele Pgi-la was found only inthe Tawu population, whereas Pgi-lb was observedin both populations. Two genotypes, ab and bb, havebeen observed so far. The proportion of polymor-phic loci, percentage of heterozygous loci per indivi-dual, the mean number of alleles per locus, and theeffective number of alleles per locus were 5 per cent,0.9 per cent, 1.05, and 1.01, respectively, for theTawu population, whereas no variation was foundfor Tsatsayalai population (Table 3).F-statistics are listed in Table 4. The X2-test wasperformed according to the formulae of Li &Horvitz (1953). The F15 value for the Pgi-1 locus wasnegative (

5 —0.099), but the x2analysisshowed nosign
—0.099), but the x2analysisshowed nosignificant deviation from zero at the 5 per centlevel, indicating that the observed distribution ofTable 3 The percentage of polymorphic loci, thepercentage of heterozygous loci per individual, the meannumber of alleles per locus, and the effective number ofalleles per locus for each population of AmentotaxusformosanaTawuTsatsayalai% Polymorphic5.00loci*%Heterozygous0.0090loci/individualt(0.009)(0.000)Mean no. of1.051.00alleles/locust(0.05)(0.00)Effective no. of1.011.00alleles/locus*The frequency of the common allele is tSE is shown in parentheses.Table 4 Results of the y contingency test and F-statisticsfor Pgi-1 in the two populations of Amentotaxus formosana27-lcU.I.v11sr'ITu'STPgi-10.804t1—0.099—0.0470.047Average—0.099—0.0470.047tNot significant (5%).genotypes within a population was in Hardy—Wein-berg equilibrium. Treating the entire species as arandom mating unit, estimates of F11 are closer tozero than F15 for the locus surveyed. The extent ofgenetic differentiation among populations (FST) was0.047. Thus, more than 95 per cent of the geneticvariation resided within a population.When the 110 individuals originating from theTawu population were divided into four categories,based on their DBH, it was found that the youngcohort with a DBH less than 5 cm had the highestheterozygosity (H = 0.25), whereas theoldercohorts, with 15-25 cm and 25 cm DBH, gavelower values of H=0.105 and H=0.111, respect-ively (Table 5). The negative values of the fixationindex indicated that the observed distribution ofgenotypes within a category had a slight excess ofheterozygotes.RAPDanalysisThe20 random primers used in this study generateda total of 289 DNA fragments (Table 1). Sixty ofthese fragments (20.8 per cent) were polymorphic,and 229 (79.2 per cent) monomorphic. The numberThe Genetical Society of Great Britain, Heredity, 77, 388—395. LOW GENETIC VARIATION IN AMENTOTAXUS FORMOSANA 393Fig. 2 RAPD polymorphism inAmentotaxus formosana using P-14.Lanes 2—9 represent eight individualsfrom the Tsatsayalai population;lanes 11—19 represent nine individ-uals from the Tawu population; Mrepresents pGEM DNA size markers.Table 5 Genotype distribution and allele frequency at Pgi-1in the Tawu population of Amentotaxus formosanain four DBH classesDBH(cm)No.5405—454215—2519�259Mean*Genotypeaaabbb0103009330217018Alleleab0.1250.8750.1070.8930.0530.9470.0560.9440.1000.900H0H,Ft0.2500.222—0.1430.2140.194—0.1200.1050.102—0.0560.1110.111—0.0590.2000.181*weighted by the number of individuals.tFixation index.of scorable RAPD fragments generated per primervaried between five and 27, while the number ofpolymorphic bands per primer ranged between oneand 19 (Fig. 2). The size of the DNA fragmentsranged between 300—3000 bp. Seven of the primers(i.e. OPE-1; UPS-i; OPY-7; OPY-iO; P-b; P-il;and P-i3) detected no variation and the mono-morphic profiles they amplified were shared by allindividuals in both populations.Observing the pairwise similarity coefficient (SC)across all possible comparisons, the maximum valueof SC (0.992) was found within the Tawu popula-tion, and the minimum value (0.939) between thetwo populations. The average similarity coefficientswithin the Tawu and Tsatsayalai populations andbetween them were 0.974, 0.970 and 0.966, respect-ively (Table 6). The between-population similarity(S,), corrected for within-populati

6 on similarity, wasThe Genetical Society
on similarity, wasThe Genetical Society of Great Britain, Heredity, 77, 388—395.0.994, and the corresponding geneticbetween the two populations was 0.006.DiscussiondistanceThe low genetic diversity detected in A. formosanaduring this study is most likely the result of thegeological history of Taiwan. Strong tectonic activi-ties (Penglai orogeny) were recorded in the middleof the Pleistocene (Teng, 1987). Several drasticvegetational changes were recorded in Taiwanduring the Pleistocene and the last 60000 years(Tsukada, 1967). The coldest climate prevailed inthe Tali glacial age or early Würm glacial age, whena rapid expansion of the boreal elements took place(Tsukada, 1966). It is hypothesized that there was adrastic reduction in the number of trees in Taiwanduring this geological age, forming a bottleneck thatM 2 3 4 5 6 7 8 9 M 11 12 13 14 15 16 17 18 19 M2645 bp1605 bp1198bp676 bp517bp460 bp396 bp250 bp 394 C.-T. WANG ETAL.Table 6 Average similarity coefficients (SC) within andbetween populations of Amentotaxus form osanaWithin populationBetween populationsTawu Tsatsayalai Not corrected CorrectedSimilarity* 0.9740.9700.9660.994(0.010)(0.012)(0.007)*SE is shown in parentheses.resulted in low genetic variation. Species such as A.formosana probably survived the extreme climatefluctuation by migrating to lower-elevation refugiaduring the Quaternary (Li, 1955). The geologicalreason for the genetic depauperation of A. formo-sana may be similar to that which caused the lowgenetic diversity in red pine; this low diversityresulted from passage through a genetic bottleneckduring glacial episodes of the Holocene (Fowler &Morris, 1977; Simon et al., 1986). The low geneticdiversity could also be a result of the small popula-tions of A. fomiosana confined to southern Taiwan.Because random genetic drift occurs particularly insmall populations (Hartl, 1980), it results in fixationof alleles after many generations.Genetic heterogeneity is often attributed to alocaladaptationto environmental variations(Hamrick et al., 1992). The FST value indicates that4.7 per cent of the genetic diversity found in thisstudy occurred between the two populations. Thislow interpopulational differentiation is consistentwith data from many other conifers (Hamrick et al.,1992).The slight but not significant excess of hetero-zygotes in the Tawu population (F15 =—0.099)isprobably because no individuals with genotype Pgi-laa were found. Indeed, Pgi-laa is probably absentfrom the whole population as a total of 110 individ-uals, which comprises approx. 16 per cent of theTawu population, was screened. However, as statedin 'Materials and methods', sampling was not abso-lutely random owing to inaccessibility. The chance ofallele Pgi-]a not being picked up was alwayspossible, given that it is a rare allele. Also the possi-bility exists that the apparent absence of the allele inthe Tsatsayalai population is the result of samplesize.The increase in frequency of allele Pgi-la in indi-viduals with decreasing DBH (Table 5) may haveseveral explanations. First, the allele Pgi-la may belost in the Tsatsayalai population but has remainedunfixed in the Tawu population; this could haveresulted from random genetic drift occurring in asmall population. Alternatively, limiting ecologicalfactors, including altitude, moisture, microclimateand their interactions found in natural habitats,suggest a

7 strong selection pressure against Pgi-l
strong selection pressure against Pgi-laa.However, these two hypotheses do not account forthe absence of the allele Pgi-la in the Tsatsayalaipopulation, which could be considered as a singlepanmictic unit with the Tawu population. Amento-taxus formosana was occasionally found betweenthese two populations even if it is uncommon. Asoutcrossing wind-pollinated gymnosperms have theleast variation among populations (Hamrick &Godt, 1990), the exchange of gametes between thesetwo populations is always possible. The occurrenceof the allele Pgi-la may also be the result of a recentmutation in the Tawu population. The frequency ofallele Pgi-la increased from 0.056 in older trees to0.125 in the youngest trees. This observation tendsto support this hypothesis, as it may explain theabsence of allele Pgi-la in the Tsatsayalai popula-tion. The increase in frequency of allele Pgi-la mayhave been caused by selection acting directly upontrees carrying this allele. The allele Pgi-la may even-tually be detected in the Tsatsayalai population, asno barrier has been found between them. However,dispersion of this allele may be slow because of thelarge and heavy seeds, which fall around the mothertree. The flowers may receive predominantly thepollen from nearby relatives, even though the pollencould also be transferred a long distance by wind.The percentage of polymorphism detected usingRAPDs was greater than that for isozyme markers.Unfortunately, no RAPD marker specific to eitherthe Tawu or the Tsatsayalai population was found.However, the lack of variation between individualswithin and between populations, as revealed byRAPD analysis, agrees with the low level foundusing isozymes. A similar observation, based onisozyme and RAPD data has been reported for redpine (Mosseler et al., 1992).AcknowledgementsWewould like to thank Prof. Shong Huang(National Taiwan Normal University) for his helpfuladvice and for stimulating discussion. This researchwas supported by grant 84AST-2,3-FOD-04 from theCouncil of Agriculture, Taiwan.ReferencesCHELIAK,W. M. AND PITEL, J. A. 1984. Techniques forstarch gel electrophoresis of enzymes from forest treeThe Genetical Society of Great Britain, Heredity, 77, 388—395. LOW GENETIC VARIATION IN AMEN TO TA XUS FORMOSA NA 395species. Can. Forestry Sen.'. Inf Rep. PI-X-42, pp. 19—45.Petawawa National Forestry Institute.CROW,J.F.ANDKIMURA, M. 1970. An Introdution to Popu-lation Genetics Theory. Harper and ROW,NewYork.FARJON, A., PAGE, C. N. AND SCHELLEVIS, N. 1993. Apreliminary world list of threatened conifer taxa. Biodi-versityand Conservation, 2, 304—326.FERET, i'. P. 1971. Isozyme variation in Picea glauca(Moench) Voss seedlings. Silvae Genet., 20, 46—50.FOWLER, D. P. AND MORRIS, R. W. 1977. Genetic diversity inred pine: evidence for low heterozygosity. Can. I ForestRes.,7, 341—347.HAMRICK, J. L. AND GODT, M. J. W. 1990. Allozyme diversityin plant species. In: Brown, A. H. D., Clegg, M. T.,Kahler, A. L. and Weir, B. S. (eds) Plant PopulationGenetics, Breeding, and Genetic Resources, pp. 43—63.Sinauer Associates, Sunderland, MA.HAMRICK, 1. L., GODT, M. J. W. AND SI-IERMAN-BROYLE, S. L.1992. Factors influencing levels of genetic diversity inwoody plant species. New Forests, 6, 95—124.HARTL, D. L. 1980. Principles of Population Genetics.Sinauer Associates, Sunderland, MA.HEUN, M., MURPH, J. P. AND PHiLIPS, T. D. 1994. A compari-son of

8 RAPD and isozyme analyses for determinin
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