Genetic Variation of Yeddo Spruce Populations in Russia

GENETIC VARIATION OF YEDDO SPRUCE POPULATIONS IN RUSSIA

Vladimir V. Potenko & Yuri D. Knysh

Department of Genetics and Breeding, Forestry Breeding and Seed Production Center, 12 Nagornaya Str., Sosnovka, 680555 Khabarovsk Territory, Russia

Received December 12,2002; accepted April 29,2003

ABSTRACT

Genetic diversity and genetic differentiation of eight populations of Yeddo spruce (Picea jezoensis (Sieb. et Zucc.) Carr.) from the Russian Far East mainland and one from the Kamchatka Peninsula were studied analyzing allozyme variation in 20 loci. The mean number of alleles per locus (A) was 2.63, the percent of polymorphic loci (P) was 88.1 %, the observed heterozygosity (H,) was 0.18 1 and the mean value of expected heterozygosity (He)amounted to 0.189 for eight mainland populations. The values of expected heterozygosity of the northern and central mainland populations were higher than in the southern part of the natural range. Regional differences in genetic variability could be due to genetic drift in the southern populations in the middle Holocene when warmer temperatures caused an upward elevational shift of spruce populations, resulting in small, isolated populations. Only 2.4 % of the total genetic variability can be attributed to variation among mainland populations. The values of genetic variation in the Kamchatka Peninsula population were different from those in the mainland populations: A = 1.85, P = 70.0, He = 0.241. Unbiased Nei's genetic distance values (DN)were low between the mainland populations of P. jezoensis and averaged 0.006. The largest values were revealed between the mainland populations and the Kamchatka Peninsula population (at average, DN= 0.08 1) that were similar to the genetic distances observed between closely related conifer species.

Key words: Picea jezoensis, allozymes, genetic variation, genetic differentiation

INTRODUCTION tain range do not support the subdivision of P. jezoensis into a number of distinct species (FROLOV1993, Yeddo spruce, Picea jezoensis (Sieb. et Zucc.) Carr., POTEMKIN1994). Higher morphological diversity of the occurs in natural and artificial stands in Russia, China, northern Sikhote-Alin populations in comparison with Korea, and Japan. In the Russian Far East, P. jezoensis the southern Sikhote-Alin populations was also re- is distributed in the Primorski Territory, in the central vealed (FROLOV1993). Studies of allozyme variation in and southern parts of the Khabarovsk Territory, in the one of P. jezoensis populations on the Kamchatka Jewish Autonomous Region, in the Amur Territory, in Peninsula (GOMORY& PAULE1 WO), three populations the southeast of the Republic of Sakha (Yakutia), on on Sakhalin Island and one population in the Khaba- Sakhalin Island, on the southern Kuriles, and in the rovsk Territory (GONCHARENKO& POTENKO 1992, central part of the Kamchatka Peninsula. Yeddo spruce GONCHARENKO& PADUTOV 2001), and one population is a dominant species in the mountain spruce-fir forests on Shikotan Island (GONCHARENKO& PADUTOV 2001) (mainly with Manchurian fir, Abies nephrolepis have been conducted. High levels of genetic diversity (Trautv.) Maxim.) of the Russian Far East that support in the populations were revealed. Levels of genetic hydrologic and climatic conditions of the territory differentiation between Yeddo spruce and other Eur- (MAN'KO1987). In Russia, the usual Latin name for asian spruces were also described in the studies. Almost Yeddo spruce is P. ajanensis. all previously analyzed populations are geographically Various studies of morphological and genetic isolated and represented marginal parts of Yeddo diversity of Yeddo spruce were conducted. Four spruce spruce natural range. However, assessments of range- species (P. kamtchatkensis Lacas., P. ajanensis Fish., wide allozyme variation of Yeddo spruce from the P. microsperma (Lindl.) Cam. and P. komarovii V. mainland areas have not been reported. Therefore, the Vassil.) were described on the basis of morphological aim of this study was to investigate levels of genetic traits (KOMAROV1934, VASIL'EV 1950) within the variation and differentiation of the nine Yeddo spruce Russian part of Yeddo spruce natural range. However, populations representing a range-wide collection from recent results of morphological diversity research in the mainland and the Kamchatka Peninsula. native Russian populations on the Sikhote-Alin moun-

O ARBORA PUBLISHERS V. V. POTENKO& Yu. D. KNYSH:GENETIC VARIATION OF PICEAJEZOENSIS POPULATIONS IN RUSSIA

MATERIALS AND METHODS

Seeds for electrophoresis were collected in nine native populations of Yeddo spruce (Fig. 1). In eight populations seeds were collected from 282 individual trees. A seed lot collected from a native population by state forest farm for artificial reforestation provided sample from the Kamchatka Peninsula. Table 1 displays the data on the population locations and the number of trees analyzed. Six megagametophytes per tree or 94 megagametophytes per seed lot were subjected to horizontal starch gel electrophoresis. Details of laboratory procedures were described in POTENKO& VELIKOV(1998). Seed tissues were analyzed for 14 enzyme systems (abbrevia- tions and EC number in parentheses): aspartate aminotransferase (AAT, 2.6.1. I), alcohol dehydrogenase (ADH, 1.1.1. l), fluorescent esterase (Fl-EST, 3.1.1. I), glucose-6-phosphate isomerase (GPI, 5.3.1.9), glutamate dehydrogenase (GDH, 1.4.1.3), hexokinase (HK, 2.7.1. I), isocitrate dehydrogenase (IDH, 1.1.- 1.42), leucine aminopeptidase (LAP, 3.4.11. I), malate dehydrogenase (MDH, 1.1.1.37), malic enzyme (ME, 1.1.1.40), phosphoglucomutase (PGM, 2.7.5. I), 6- phosphogluconate dehydrogenase (6-PGD, 1.1.1.44), Figure 1. Range of P. jezoensis (MAN'KO1987) and the shikimate dehydrogenase (SkDH, 1.1.1.25) and sorbitol locations of nine populations sampled for electrophoretic dehydrogenase (SDH, 1.1.1.14). Enzyme staining analysis followed standard methods (CHELIAK& PITEL1984) with non-significant modifications. Inheritance of electrophoretic enzyme variants of Yeddo spruce was and non-simultaneously (A~t-2'.~"and A~t-2~).~") determined earlier (GONCHARENKO& PADUTOV 200 1). migrating variants (Fig. 2). No significant deviations Some attention must be paid to inheritance of from the expected 1: 1 segregation ratio were observed enzyme variants attributed to the Aat-2 zone. Unlike in any heterozygous tree with simultaneously or non- the other Eurasian spruces (LUNDKVIST1979, POULSEN simultaneously migrating bands at the Aat-2 zone et al. 1983, MUONAet al. 1987, GONCHARENKO& tested by Chi-square criterion (P < 0.001). Segregation PADUTOV2001, LEWANDOWSKIet al. 2002), the two- patterns were received from 16 and 22 megagameto- banded AAT-2 zone of Yeddo spruce has both simulta- phytes of two heterozygous trees with non-simultaneously (Aat-2°.65,A~t-2'.'~, Aat-2'.15, and at-2'.17) neously migrating bands in combination Aat-2'.LX)".20

Table 1. Location of the sampled population and number of analyzed trees.

Latitude Longitude Number Population / Location (" N) (" E) of trees

12 Khorogochi (Amur Territory) 34 Dipkun (Amur Territory) 56 Snezhnyi (Khabarovsk Territory) 78 Burga (Khabarovsk Territory) 9 Tukhala (Khabarovsk Territory) Khekhtsir (Khabarovsk Territory) Dolmi (Khabarovsk Territory) Uglekamensk (Primorski Territory) Atlasovo (Kamtchatka Peninsula)"

"ulked seed lot analyzed. Total weight of seed lot is 50 kg. ' Number of analyzed seeds per seed lot. Allele Pre-mRNA transcripts Alternative mRNAs Polypeptide Electrophoregram of AAT-2 zone of splicing variants megagarnetophytesfrom P. jezoensir heterozygote trees Aat-2u51Aal-21m and ~i-3.~IAO~-2'"' with simultaneous and non-simultaneous segregation, respectively

Figure 2. Generalized schemes of two types (A and B) of alternative splicing of pre-mRNAs transcribed from a single enzyme- coding gene (modified from MANCHENKO2001) that explain the enzyme electrophoregram phenotypes of megagametophytes of P. jezoensis with simultaneously and non-simultaneously migrating bands of Aat-2 zone. Introns and exons are numbered and shown by light (0 0)and dark boxes (I I I I), respectively. The pre-mRNA transcripts are spliced (X)in two alternative ways. Vertical arrows (ti) indicate sites of alternative splicing. Exons containing nucleotide substitutions responsible for different allozyme variations are marked by different number of asterisks (*, **). Allelic variants of polypeptides are designated a', a" and p', respectively. and Aat -2 1 .(M/O.8O respectively. A possible explanation with frequency more than 5 % per locus (A,,) was is that the two-banded Aat-2 isozymes are the result of calculated. Fixation indices (FIs, FITand FsT) were used alternative splicing. Alternative splicing is a mechanism for assaying the population genetic structure (NEI where parts of the pre-rnRNA are either excluded from 1977). Calculations of the F-statistics, observed or included in the mature rnRNA. It allows different heterozygosity (H,) and chi-square tests of homogeneity protein isoforms to be created from a single gene. At for allele frequency variation among populations were the single gene level, nucleotide substitutions in exon performed for the eight populations where individual not subjected to alternative splicing is expressed in tree seed collections were conducted (Table 1). NEI'S simultaneously migrating protein isoforms (Fig. 2, A), unbiased genetic distances (NEI 1978) were calculated. while nucleotide substitutions in exon subjected to The relationships among the populations were visual- alternative splicing is expressed in non-simultaneously ized by constructing of phylogenetic tree by unweighted migrating protein isoforms (Fig. 2, B) (SMITHet al. pair-group method with arithmetic means (UPGMA), 1989, MANCHENKO2001). In this case simultaneously using the genetic distances and bootstrap estimates to and non-simultaneously migrating variants at the Aat-2 test the reliability of the tree using the DISPAN com- zone can be considered as products of different alleles puter program (OTA1993). of single gene. It may be noted that in humans about 30% of all pre-mRNA transcripts are subject to alternative splicing (GELFANDet al. 1999). RESULTS Allele frequencies were analyzed using the BIOSYS- 1 computer program (SWOFFORD& SELANDER Staining of the 14 enzymes revealed gene products of 1989). For each population, the mean number of alleles 20 loci. Without exception, variant alleles were found per locus (A), percentage of polymorphic loci (P and at every locus (Table 2). Most polymorphic loci were P,), observed (H,) and expected heterozygosity (He) Aat-2, Idh-1, Sdh (6 alleles at each locus) and Idh-2, were computed. In addition, the mean number of alleles Mdh-3 and Hk (5 alleles at each locus). The mainland

O ARBORA PUBLISHERS V. V. POTENKO& Yu. D.~YSH: GENETIC VARIATION OF PICEAJEZOENSIS POPULATIONS IN RUSSIA

Table 2. Allele frequencies in the natural populations of P. jezoensis.

- - Population Locust Khorogochi Dipkun Snezhnyi Burga Tukhala Khekhtsir Dolrni Uglekarnensk Atlasovo

Aat-1 0.90 1 .oo 1.05 1.10 Aat-2 0.65 0.80 1.oo 1.15 1.I7 1.20 Adh 0.90 1.oo 1.10 Fl-Es~ 0 0.70 1.oo 1.30 Gdh 0.65 0.80 1.oo 1.35 Gpi 0.80 1 .oo 1.20 Hk 0.80 0.90 1.oo 1.10 1.20 Idh -1 0 0.90 1.oo 1.05 1.10 1.15 Idh -2 0.70 0.90 1.oo 1.O5 1.20

populations had quite similar allele frequencies. How- allele frequencies between the mainland populations ever, the Aat-1, Aat-2, Adh, Idh-2, Lap-I, Mdh-3, and the Atlasovo population from the Kamchatka Pgm-2 and Skdh-I loci showed some differences in Peninsula. FORESTGENETICS 1O(1): 55-64,2003

Table 2. (continued).

Population Locus Khorogochi Dipkun Snezhnyi Burga Tukhala Khekhtsir Dolrni Uglekamensk Atlasovo

Lap -1 0 0.94 1 .oo 1 .O4 Lap -2 0.95 1 .oo 1.05 Mdh -1 0.90 1.oo 1.14 Mdh -2 0.80 1 .oo 1.20 Mdh-3 0 0.45 0.75 0.90 1.oo Me 0.20 1.oo 1.65 2.00 6-Pgd-1 0.90 1.oo 1.10 Pgm -1 0.90 0.95 1 .oo 1.10 Pgm -2 0.85 1 .oo 1.15 Scfh 0 0.95 1 .oo 1.05 1.10 1.15 Skdh 0.95 1.oo 1.05 1.20

O ARBORA PUBLISHERS V. V. POTENKO& Yu. D.KNYSH: GENETIC VARIATION OF PICEAJEZOENSIS POPULATIONS IN RUSSIA

Table 3. Genetic variability at 20 loci in 11 populations of P. jezoensis (standard errors in parentheses).

Population

Khorogochi Dipkun Snezhnyi Burga Tukhala Khekhtsir Dolmi Uglekamensk Mean for 8 mainland populations

Atlasovo

a Unbiased estimate (see NEI, 1978)

Table 4. F-statistics for 20 polymorphic loci and test of homogeneity of allele frequency.

Locus

Aat-1 Aat-2 Adh Fl-Est Gdh Gpi Hk Idh -1 Idh -2 Lap -1 Lap -2 Mdh -1 Mdh -2 Mdh -3 Me 6-Pgd -1 Pgm -1 Pgm -2 Sdh Skdh

Mean 0.007 0.03 0.024 20.4 19 2 1 ns

Parameters of genetic variation were calculated on 0.145 to 0.211. The values of expected heterozygosity the basis of frequencies of 82 alleles of 20 loci. For the of the northern and central mainland populations were eight mainland populations, the mean number of alleles higher than in the southern part of the natural range. per locus ranged from 2.20 to 3.05, and averaged 2.63. The values of genetic variation in the Atlasovo popula- The percent of polymorphic loci (P) ranged from 65.0 tion were different from those for the mainland popula- to 95.0%, with an average of 88.1%. The observed tions and reached: A = 1.85, P = 70.0, He = 0.241 heterozygosity was from 0.136 to 0.207, with an (Table 3). average of 0.181. The mean value of expected FIS values ranged from -0.069 for Hk to 0.126 for heterozygosity amounted to 0.189, with variation from Adh, with an overall mean of 0.007. F,, values at the Table 5. Estimates of NEI'S (1978) genetic distances.

Population 1 2

Khorogochi Dipkun Snezhnyi Burga Tukhala Khekhtsir Dolmi Uglekamensk Atlasovo

Aat-2, F1-Est, Hk, Iclh-I, and Lap-2 loci were negative Atlasovo population from the Kamchatka Peninsula is and at 15 loci were positive, reaching 0.191 at A&. rather distant from these clusters. Mean FITwas 0.030. Positive FIs and FITvalues indicate a slight deficiency of heterozygotes relative to the Hardy-Weinberg equilibrium for the populations DISCUSSION investigated and for I? jezoensis in total. The FsT value at 20 loci was 0.024. Thus, about 98% of the total P. jezoensis exhibited levels of genetic variation that genetic variability resided within populations and only were similar to those reported for predominantly 2.4% among populations (Table 4). outcrossed, wind-pollinated species with a regional Chi-square tests of homogeneity for allele frequen- distribution (HAMRICKet al. 1992). Genetic diversity cies in the mainland populations showed that there are maintained by the mainland populations of I? jezoensis no significant differences at most of the loci. Among (He = 0.189) was slightly higher than the average value the populations the most heterogenous allele frequen- for a number of spruce species analyzed at more than cies were revealed at the Adz, Me and 6-Pgd-I loci 18 loci (H, = 0.180) (GONCHARENKO& PADUTOV (Tdble 4). 2001). Our estimates for Yeddo spruce were higher Unbiased Nei's genetic distance values (D,) were than thosc for I? engel~nannii(Parry) Engelm. (He = low between the mainland populations (0.000-0.026) cf 0.152, SHEA1990), P. glauca (Moench.) Voss. (He = I? jezoensis and averaged 0.006. The lergest values 0.174, YEI-I& AKNOTT1986), l? ~lariana(Mill.) B. S. (0.077-0.084, at average, DN= 0.08 1) wcre detected P. (He = 0.107, YEI-Iet al. 1986), P. omorika (Pang.) between these populations and the Atlasovo population Purk. (He = 0.130, KUITTINENet al. 1991), l? rubens (Table 5). Sarg. (He = 0.079, HAWLEY& DEHAYES1994), and P. UPGMA cluster analysis (Fig. 3) split the mainland sitchensis (Bong.) Carr. (He = 0.150, YEH & populations into two clusters, a c!uster of the central EL-KASSABY1%HI). The Atlasovo population exhibited and northern populations and a cluster of the southern a much higher level of genetic variation (It = 0.241), populations. These clusters on the phylogenetic tree similar to those reported for widespread Eurasian were confirmed by bootstrap values. The marginal spruces such as I? abies (L.) Karst. and I? obovata Khorogochi population is close to these clusters. The Ledeb. (He = 0.252 and He = 0.213, respectively, KIWTOVSKII8t BERGMANN 1995, H, = 0.193 and H, = 0.257, respectively, GONCHARENKO&PADUTOV 200 1). Khorogochi Dipkun The values of expected heterozygosity of the Snezhnyi norlhern and central mainland populaiions are higher Tukhala Burga than those of the southern ones, as for red spruce Khekhtsir & Dolmi (HAWLEY DEHAYES1994). It is possible that the Uglekarnensk higher genetic variability evident in Yeddo spruce from Atlasovo the northern 2nd central mainland regions and lower genetic variability in the south could reflect differences resulting from past migrating patterns in the late Pleis- Figure 3. Dendrogram of UPGMA clusler analysis based on tocene - Holocene. Studies of fossil conifer pollen NEI'S (1978) genetic distances betwecn the ninc populations (KOROTKIIet a!. 1997) indicate that 18-20 thousand of Yeddo spruce. Numbers within the phylogenctic tree years ago the vegetation of the Sikhote Alin was similar represent bootstrap ilumbcrs bascd on 1000 rcp1ica:es. V. V. POTENKO& Yu. D. KNYSH:GENETIC VARIATION OF PICEAJEZOENSIS POPULATIONS IN RUSSIA to that of the contemporary north-west coast of the Sea PADUTOV200 1). of Okhotsk. The latitudinal shift to the south was The rapid expansion of Yeddo spruce to the north almost 10". Thus, the modern counterpart of natural in the late Pleistocene can explain the low level of vegetation for the south Sikhote-Alin in that time is interpopulation differentiation between the mainland now roughly situated at the northern limit of the I? populations (FsT = 0.024), and it is similar to the jezoensis natural range where it forms isolated stands average value for the island populations of I? jezoensis (MAN'KO1987). After the climate had cooled Yeddo (FsT = 0.030) (GONCHARENKO& PADUTOV2001), spruce expanded into northern Sikhote-Alin, where in although lower than in several other spruce species (GsT the middle Holocene period a zone of Korean pine- = 0.055) (HAMRICKet al. 1992). spruce-broadleaf and fir-spruce mixed forests formed. Low estimates of NEI'S genetic distances confirm During this period in the south Sikhote-Alin, the close genetic relationship between most of the warmer temperatures caused an upward elevational mainland populations (DN= 0.006). The genetic drift shift of the Yeddo spruce populations to the altitude occurring in the isolated Khorogochi population could about 1500 meters above sea level. The rapid popula- explain the higher level of differentiation from the other tion growth in the south Sikhote-Alin in altitudes mainland populations (DN= 0.015). The mean value of between 700 and 1200 meters above sea level was a genetic differentiation was slightly lower than those result of a decrease in temperatures and an increase of between the five populations of Yeddo spruce from the relative humidity in the late Holocene (GOLUBEVA& mainland, Sakhalin Island and Shikotan Island (DN= KARAULOVA1983, KOROTKIIet al. 1997). 0.009-0.01 2) studied earlier (GONCHARENKO& POTEN- Levels of genetic variability may have been reduced KO 1992, GONCHARENKO& PADUTOV2001). The in the south where warmer temperatures caused an largest value (DN= 0.081) was revealed between the upward elevational shift of the spruce populations, mainland populations and the Atlasovo population from resulting in small, isolated populations that were the Kamchatka Peninsula. This should not be consid- possibly subjected to increased levels of inbreeding and ered as biased sampling from the Kamchatka Peninsula, increased genetic drift. Theoretical studies of the because similar allele frequencies were found in six loci genetic effects of population bottlenecks have shown (Gdh, Aat-1, Aat-2, Lap-1, Lap-2 and Mdh-1) studied that if the population expands rapidly after a bottleneck, here and in the work of GOMORY& PP.ULE(1990). an increased number of rare alleles due to new muta- In 1934 KOMAROV(1934) defined the taxonomic tions will be found until equilibrium heterozygosity has status of several stands in the Kamchatka Peninsula as been attained, with the discrepancy being most pro- a separate species I? kamtchatkensis Lakas. Later, nounced for rare alleles (MARUYAMA& FUERST 1984). VASIL'EV(1950) subdivided P. jezoensis into three This can explain higher values of the mean number of species: f? ajanenesis Fish., I? microsperma (Lindl.) alleles per locus in the most mainland population (A = Carr. and f? komarovii V. Vassil., but the Kamchatka 2.63) in comparison with Sakhalin Island (A = 2.21) Peninsula stands were described as I? ajanenesis (GONCHARENKO& PADUTOV 200 1) and the Kamchatka populations isolated from the main range. Independent Peninsula (A = 1.85) populations that were likely to be taxonomic status of I? ajanenesis, I? microsperma and not subjected to fluctuation of population during the I? komarovii was widely accepted (VOROB'EV1968, Pleistocene and Holocene periods. The relatively low USENKO1969, U~usov1995). However, recent re- value of the mean number of alleles per locus (A = search on the morphological diversity in the native 2.20), the high level of expected heterozygosity (He = Russian populations on the Sikhote-Alin mountain 0.21 1) and the mean number of unrare alleles per locus range does not support the subdivision of f? jezoensis (A,, = 1.85) in the Khorogochi population may be a into a number of distinct species (FROLOV1993, result of genetic drift occurring in a quite small popula- POTEMKIN1994). tion (< 100 trees) isolated from the other mainland According to the descriptions of the species ranges populations. Loss of alleles occurs more rapidly than made by VASIL'EV(1950), and U~usov(1995) the loss of heterozygosity after severe restrictions in the Khorogochi, Dipkun and Snezhnyi populations repre- population size as shown in studies of MARUYAMA& sent f? ajanenesis, Burga, Tukhala, Khekhtsir and FUERST(1 985). Dolmi populations - I? microsperma, and Ugleka- Positive FIsand FITvalues indicate that deficiency mensk population - I? komarovii. The low genetic of heterozygotes was low for the populations investi- differentiation between those mainland populations gated and for I? jezoensis in total. It should be noted observed in this study does not support the subdivision that an excess of heterozygotes was found in the of I? jesoensis. Meanwhile, higher genetic differentia- Sakhalin Island and Shikotan Island populations tion between the mainland populations and the Atlaso- (GONCHARENKO& POTENKO 1992, GONCHARENKO& vo population, the existence of a clear range border of spruce in Kamchatka Peninsula and the absence of USSR Far East South]. Nauka, Moskow, 143 pp. [In introgressive hybridization between mainland and Russian] Kamchatka's populations allowed us to consider the GOMORY,D. & PAULE,L. 1990: [Comparison of the genetic spruces from mainland and Kamchatka Peninsula as variability of the spruce populations growing on the territory of the USSR and Slovakia.] In: Intensification of distinct taxa. The observed level of genetic differentia- management in spruce stands in the context of ecology tion between the mainland and the Kamchatka popula- conditions. pp. 2-1 2. Vysoka Skola Lesnicka a Drevarska tions was similar to those between closely related vo Zvolene, Zvolen, Slovakia. [In Russian] coniferous species such as Picea abies and P. obovata GONCHARENKO,G G & PADUTOV,V. E. 2001 : [Population (DN= 0.072, KRUTOVSKII& BERGMANN1995, DN= and evolutionary genetics of spruces of the Palaearctic]. 0.083, GONCHARENKO& PADUTOV200 1), Pinus IL NAN B, Gomel, 197 pp. [In Russian] virginiana Mill. and P. clausa (Champ. ex Engelm.) GONCHARENKO,G G & POTENKO,V. V. 1992: [Genetic diversity and differentiation of Picea ajanenesis Fisch. in Vasey ex Sarg. (DN= 0.07 1, PARKERet al. 1997), and natural populations of Sakhalin Island and south of taeda L. and I? echinata Mill. (DN = 0.074, P. Khabarovsk Territory]. Proc. of Russian Acad. Sci: 325: EDWARDS-BURKEet al. 1997). 838-844. [In Russian] The time of geographic isolation of the Kamchatka HAMRICK,J. L., GODT,M. J. W. & SHERMAN-BROYLES,S. L. Peninsula vegetation since the mid-Quaternary 1992: Factors influencing levels of genetic diversity in (KOLESNIKOV1961) is in accordance with the time woody plant species. New Forests 6: 95-1 24. calculated on the basis of the mean DNvalue between HAWLEY,G J. & DEHAYES,D. H. 1994: Genetic diversity and the mainland and the Kamchatka Peninsula populations population structure of red spruce (Picea rubens). Can. J. (t = 405,000 years) using the NEI'S(1975) equation: t = Bot. 72: 1778- 1786. 5 xlOhD,. KOLESNIKOV,B. P. 196 1: [Vegetation. In: Fundament of the Far East.] (Ed. G D. Rihter). pp. 183-245. Acad. Sci. Further studies of genetic differentiation between USSR Publishing House, Moskow. [In Russian] the mainland population of Yeddo spruce and the KOMAROV,V. L. 1934: [Flora of the USSR.]. Acad. Sci. population of I? jezoensis var. hondoensis (Mayr) USSR Publishing House, Leningrad. 302 pp. [In Russian] Rehd. from Honshu Island (Hondo spruce) would make KOROTKII,A. M., GREBENNIKOVA,T. A., PUSHKAR,V. S., a considerable contribution to the investigation of the RAZJIGAEVA,N. G, VOLKOV,V. G, GANZEY,L. A., species. MOKHOVA,L. M., BAZAROVA,V. B. & MAKAROVA,T. 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