Genetic diversity and population structure ofKorean alder (Alnus japonica; Betulaceae)
Man Kyu Huh
Abstract: The genetic diversity and population genetic structure ofAlnus japonica(Thunb.) Steudel in Korea werestudied and compared with those of alder from Canada. Nineteen of the 25 loci studied (76.0%) showed detectablepolymorphism. The mean genetic diversity within populations was 0.207, which was higher than that for two Canadianalder species (Alnus rugosa(Du Roi) Spreng. andAlnus crispa(Ait.) Pursh). Analysis of fixation indices, calculatedfor all polymorphic loci in each population, showed a substantial deficiency of heterozygotes relative to HardyWeinberg expectations. The mean population differentiation value ofA. japonica in Korea (GST = 0.095) is similar tothose ofA. rugosain Canada (GST = 0.052). These low values ofGST in two countries, reflecting little spatial geneticdifferentiation, may indicate extensive gene flow (via pollen and (or) seeds) and (or) recent colonization.
Rsum: Lauteur a tudi et compar la diversit gntique et la structure gntique des populations dAlnus japonica(Thunb.) Steudel en Core celles de laulne au Canada. Parmi les 25 loci tudis, 19 (76,0%) affichaient unpolymorphisme dtectable. La diversit gntique moyenne au sein des populations tait de 0,207. Cette diversit taitplus leve que celle de deux espces canadiennes daulne (Alnus rugosa(Du Roi) Spreng. etAlnus crispa(Ait.)Pursh). Lanalyse des indices de fixation, tels questims pour tous les loci polymorphes au sein de chaque population,indiquait une dficience substantielle en htrozygotes comparativement aux proportions espres selon lquilibredHardyWeinberg. La valeur moyenne de diffrenciation des populations chez lA. japonicaen Core (GST = 0,095)tait similaire celles de lA. rugosaau Canada (GST = 0,052). Ces faibles valeurs deGST au sein des deux paysrefltent un diffrenciation gntique spatiale faible, laissant supposer un flux gnique important (par pollen ou pargraines) et (ou) une colonisation rcente.
[Traduit par la Rdaction] Huh 1316
IntroductionAlder is an early successional monoecious species that
forms root nodules symbiotically with the actinomyceteFrankia, which fixes nitrogen (Normand and Lalonde 1986).As revealed by early fossil record, the genusAlnus couldhave originated in the Asian land mass, around the Creta-ceous (Furlow 1979a). Alder is now distributed in Asia,Southeast Asia, and the East Indies and the species is alsofound in subtropical and tropical New World north to thesouthern United States (Woodland 1991). Speckled alder(Alnus rugosa(Du Roi) Spreng.) and green alder (Alnuscrispa (Ait.) Pursh) also occur in the region of central Que-bec in North America (Bousquet et al. 1988). It is assumedthat the differentiation of the alder species preceded thespread of the genus to the Northern Hemisphere, but thisevent has not been documented precisely in time (Furlow1979a). Fossil records suggest that species of both subgeneraexisted by the Miocene (ca. 20 million years B.P.) in NorthAmerica (Furlow 1979a).
This study investigated the genetic diversity of Korean al-der populations. It will be of interest to analyze one of theprogenitor populations, widespread plants from Korean al-der, on the amounts and patterns of genetic variation. Littleis known about the levels of genetic variation and the popu-
lation structure of alder species, despite its ecological impor-tance (furniture, forestation, firewood, and windbreak forest)and transcontinental distribution of the species. This actino-rhizal wind-pollinated tree is typically found on moist andsoggy lowland soil; it reaches a height of 10 m and maxi-mum expected stem age of 20 years, although individualsmay be actually older because of root and stump sprouting(Huenneke 1987).
Alnus rugosaand A. crispa are classified by Furlow(1979a, 1979b) as subspecies ofAlnus incana(L.) Moench(Alnus incana ssp. rugosa (Du Roi) Clausen andAlnusincanassp.crispa (Ait.) Pursh), while both are members ofsubgenusAlnus(Furlow 1979a). Bousquet et al. (1988) sug-gest that alder originated in Asia and India. Actually, Asianregions such as China, Korea, Japan, and Siberia are wellknown for various alder species. The genusAlnus in Koreais comprised of 15 species.Alnus japonica(Thunb.) Steudelis the most abundant alder species in Korea. Female flowersof alder consist of two compound, united carpels. The spe-cies is obligating outcrossing with wind pollination. Iwanted to address the following questions in this study: isthere considerably more variability in the putative area oforigin of the genusAlnusand how extensive does the loss indiversity concur with speciation, adaptation, and the spreadto different climate regions or to different continent? Thepurpose of this study was (i) to estimate how total allozymediversity is maintained inA. japonicaand (ii ) to compare thegenetic diversity and structure of alder in Korea with thoseof alder from Canada.
Can. J. For. Res.29: 13111316 (1999) 1999 NRC Canada
Received August 8, 1998. Accepted February 11, 1999.
M.K. Huh. Department of Biology Education, Pusan NationalUniversity, Pusan, 609-735, The Republic of Korea.e-mail: email@example.com
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Materials and methods
Sampling procedureAlnus japonicawas collected from 17 natural populations in Ko-
rea (Fig. 1). One leaf per plant was sampled during 19961998.More than 30 plants were collected from each population. Leavesgathered from natural populations were stored in plastic bags forseveral days in a refrigerator until electrophoresis was carried out.
Enzyme electrophoresisLeaves were homogenized by mechanical grinding to release en-
zymes from cell and organellar membranes with Tris-HCl grindingbuffer polyvinylpyrrolidone solution described in Soltis et al.(1983). Electrophoresis was performed using 10% starch gel.Tris citric acid electrophoretic buffer system (No. 5 of Soltis etal. 1983) was used for isocitrate dehydrogenase (IDH), malatedehydrogenase (MDH), malic enzyme (ME), 6-phosphoglugonatedehydrogenase (6PGD), phosphoglucomutase (PGM), shikimatedehydrogenase (SKD), and superoxide dehydrogenase (SOD); asodium borate system (No. 6 of Soltis et al. 1983) was used forglutamate oxaloacetate transaminase (GOT) and phosphoglucoseisomerase (PGI); a lithium borate system (No. 7 of Soltis et al.1983) was used for menadione reductase (MNR); and a morpholinecitrate pH 6.3 buffer (Werth 1991) was used for fluorescentesterase (FE) and peroxidase (PER). Presumptive loci were desig-nated sequentially, with the most anodally migrating one desig-nated No. 1; the next, No. 2; and so on. Likewise, alleles weredesignated sequentially with the most anodally migrating one des-ignated a and progressively slower forms b, c, and so on. AllA. japonicaisozymes expressed phenotypes that were consistent insubunit structure and genetic interpretation with most isozymestudies in plants, as documented by Weeden and Wendel (1989).
Analysis of dataA locus was considered polymorphic when more than one allele
was detected, regardless of their frequencies. Four standard geneticparameters were estimated using a computer program developedby M.D. Loveless and A. Schnabel (personal communication): per-centage of polymorphic loci (P), the number of alleles per poly-morphic locus (AP), mean number of alleles per locus (A), effectivenumber of alleles per locus (AE), and gene diversity (HE) (Hamricket al. 1992). Subscripts refer to species- (s) or population-level (p)parameters. Observed heterozygosity (HO) was compared withHardyWeinberg expected value using Wrights fixation index (F)or inbreeding coefficients (Wright 1922). These indices were testedfor deviation from zero by2 statistics following Li and Horvitz(1953).
Neis gene diversity formulae (HT, HS, DST, andGST) were usedto evaluate the distribution of genetic diversity within and amongpopulations (Nei 1973, 1977). In addition,2 statistics were usedto detect significant differences in allele frequencies among popu-lations for each locus (Workman and Niswander 1970). Neis ge-netic identity (I) and distance (D) were calculated for eachpairwise combination of populations (Nei 1972). I used the PC-SAS program (SAS Institute Inc. 1989) to conduct a cluster analy-sis on genetic distances via the unweighted pairwise groupsmethod arithmetic average (UPGMA).
The genetic structure within and among populations was alsoevaluated using Wrights (1965)F statistics:FIT and FIS. The FITandFIS coefficients measure excesses of homozygotes or heterozy-gotes relative to the panmictic expectations within the entire sam-ples and within populations, respectively. TheGST coefficientestimates relative population differentiation. Deviation ofFIT andFIS from zero was tested using2 statistics (Li and Horvitz 1953).Two indirect estimates of gene flow were calculated. One estimateof Nm (the number of migrants per generation) was based onGST
(Wright 1951) and the other estimate was based on the average fre-quency of rare alleles found in only one population (Slatkin1985; Barton and Slatkin 1986). The absolute population differen-tiation (Dm) was calculated using Neis (1973) statistics. Correla-tion between geographical and genetic distance was tested usingMantels test as advocated by Smouse et al. (1986).
Genetic diversityNineteen of the 25 loci studied (76.0%) showed detectable
polymorphism in at least two populations. The remaining sixloci (Per-3, Per-4, Mdh-3, Gpi-1, Pgm-2, and Skd-2) weremonomorphic in all populations. An average of 62.8% of theloci were polymorphic within populations, with individualpopulation values ranging from 48.0 to 72.0% (Table 1).In the polymorphic loci, 10 loci (Fe-2, Idh-1, Mdh-2, Got,Me-2, Per-2, 6Pgd-2, Sod, Pgm-1, and Skd-1) expressedthree alleles, while the remaining ones expressed either twoalleles (six loci) or four alleles (Mdh-2 andMnr). The larg-est number of alleles per locus was five atFe-1. The numberof alleles per polymorphic locus (AP) was 2.47 (2.252.88).The average number of alleles per locus (A) was 1.93 across
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1312 Can. J. For. Res. Vol. 29, 1999
Fig. 1. Location of Alnus japonicapopulations sampled forisozyme analysis and mean rainfall per year. Letters show meanannual rainfall amounts. A, 8001000 mm/year; B, 10011200 mm/year; C, 12011400 mm/year; D, 14011600 mm/year;E, 16011800 mm/year; F, 18012000 mm/year.
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populations, varying from 1.64 to 2.20. The effective num-ber of alleles per locus was similar at the species and thepopulation level (AEs = 1.40;AEp = 1.39). The mean geneticdiversity within populations was 0.207. Population 5 had thehighest expected diversity (0.282), while population 8 hadthe lowest (0.169). Genetic diversity at the species level washigh, whereas the value at the population level was low(HEs = 0.235 andHEp = 0.207, respectively).
Genetic structureIn general, genotype frequencies do not conform to
HardyWeinberg expectations. Chi-square tests indicatedsignificant deviations from HardyWeinberg ratio. As ex-pected from the chi-square tests,FIS, a measure of the devia-tion from random mating within 17 populations, was 0.422,ranging from 0.002 forGpi-2 to 0.797 for Idh-1 (Table 2).The observed significant positiveFIS value (0.422) indicatesa significant deficit of heterozygotes in the populations.
Wrights F statistics in Table 2 show that significant defi-ciencies of heterozygote exist for all polymorphic loci. On aper locus basis, the proportion of total genetic variation dueto differences among populations (GST) ranged from 0.028for Per-2 to 0.263 forFe-3 with a mean of 0.095, indicatingthat about 10% of the total allozyme variation was amongpopulations (Table 2). Thus, the majority of genetic variance(90%) resided within populations. The values of genetic dis-tance (D) were below 0.10 in most cases except in pairs in-volving the population 1. The estimate of gene flow basedon GST was relatively moderate (Nm = 2.38). Nm valuesgreater than 1 are considered high. As a result, genetic driftmay not be one of the major factors inA. japonicapopula-tions. Genetic identity values among pairs of populationsrange from 0.914 to 0.992. The genetic similarity amongA. japonicapopulations can be seen in the UPGMA dendro-
gram, where all populations clustered at a genetic distancebelow 0.095. The UPGMA dendrogram provided a few in-sights into the genetic structuring of populations (Fig. 2). Inaddition, the correlation between genetic distance and geo-graphic distance was low (r = 0.30,p < 0.05), indicating thatabout 90% of the variation in genetic distance was caused byunknown factors other than distance.
Alnus japonicain Korea maintains a higher level of allo-zyme variation (Ps = 76%, As = 2.44, andAEp = 1.40) thanmost of other long-lived, woody species, which average65% polymorphic loci (Ps), 2.22 alleles per locus (As), and1.24 effective alleles per locus (AEs) (Hamrick et al. 1992).Genetic diversity at the species level (HEs) in A. japonica(0.235) is higher than the mean for long-lived woody species(0.177) and outcrossing, wind-pollinated species (0.173).The same trend is observed at the population level too.Mean percentage of polymorphic loci (PP) for long-livedwoody perennials is 49.3%, mean number of alleles per lo-cus (AP) is 1.76, and mean effective number of alleles per lo-cus (AEp) is 1.20 (Hamrick et al. 1992). WithinA. japonicapopulationsPP is 62.8%,AP is 1.93, andAEp, 1.39.Alnus ja-ponica also maintains higher amounts of genetic diversity(HEp = 0.207) than the most gymnosperm species (meanHEp = 0.151) and long-lived, woody angiosperms (meanHEp = 0.143).
Genetic diversity ofA. japonica in Korea is comparablewith other alder species reported by Bousquet et al. (1988),although there is the difference in species (A. rugosa,A. crispa, andA. japonica) and methodology (e.g., the num-ber of loci, populations, and enzyme system, as well as sam-ple sizes) that may preclude meaningful direct comparisons
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Population N* P AP A AE HOp HEp
1 36 60.00 2.80 2.08 1.44 0.096 (0.011) 0.223 (0.046)2 38 64.00 2.88 2.20 1.59 0.118 (0.013) 0.270 (0.047)3 39 64.00 2.50 1.96 1.39 0.090 (0.011) 0.202 (0.044)4 42 68.00 2.71 2.16 1.56 0.165 (0.014) 0.273 (0.046)5 32 72.00 2.56 2.12 1.56 0.176 (0.015) 0.282 (0.045)6 30 72.00 2.28 1.92 1.33 0.148 (0.014) 0.208 (0.035)7 32 64.00 2.50 1.96 1.32 0.121 (0.012) 0.191 (0.038)8 36 64.00 2.25 1.80 1.26 0.104 (0.012) 0.169 (0.035)9 30 52.00 2.54 1.80 1.26 0.080 (0.010) 0.158 (0.038)10 38 64.00 2.31 1.84 1.35 0.112 (0.012) 0.195 (0.041)11 36 64.00 2.31 1.84 1.28 0.098 (0.011) 0.177 (0.036)12 36 68.00 2.47 2.00 1.37 0.132 (0.013) 0.203 (0.040)13 32 64.00 2.56 2.00 1.49 0.123 (0.012) 0.226 (0.046)14 30 56.00 2.29 1.72 1.32 0.109 (0....