Olga V. Nakonechnaya, Olga G. Koren, Alla B. Kholina, Yuri N. Zhuravlev POPULATION-GENETIC STRUCTURE OF A RARE PLANT ARISTOLOCHIA CONTORTA BUNGE (ARISTOLOCHIACEAE) Institute of Biology and Soil Science, FEB RAS, Vladivostok

Aristolochia contorta Bunge A. contorta Bunge is a unique herbaceous liana of the Russian Far East, where this plant is represented by a few small fragmented and badly exhausted populations in the southwest part of the Primorsky Krai and in the Jewish Autonomous Region only. In Russia, the species is listed in the Red Book of the Primorsky Krai as a vulnerable species. This plant is uncommon in nature through its sensitivity to destructive changes of habitats. Primorsky Krai

Sericinus montela Gray In addition, A. contorta is a valuable medicine plant and its collection for a herbal remedy contributes to reduction of its natural populations. Now these populations need in protection and restoring. It would be specially important for the saving not only this species, but also for the closely connected with A. contorta relict butterfly Sericinus montela Gray, for which this plant is the single feed source.

In this work we try to estimate genetic variation and differentiation of the Russian populations of A. contorta using allozyme markers. The following tasks were formulated: 1. To estimate the main parameters of genetic variation for four natural populations of A. contorta from Petrovka, Sukhodol and Razdol’naya rivers basins in Primorsky krai. 2. To estimate variation and differentiation degree among the populations.

Primorsky Krai Petrovka river Sukhodol river Razdol’naya river Fig. 1. The map of Aristolochia contorta area in Primorsky Krai

Fig. 1. Schematic representation of electrophoretic patterns of enzymes (EST, FE, PGM, GPT, IDH, GPI, SKDH, ADH, LAP, AAT, 6-PGD, ACP, ME, MDH) studied in Aristolochia contorta liaves.

Plants% P S ASAS НOНO НEНE G ST (F ST ) References Aristolochia contorta (0.019) 0.09 (0.022) Our data Rare36.7 (4.9) 1.94 (0.208) (0.022) (0.040) (Gitzendanner, Soltis, 2000) Endemic42.5 (7.6) 1.82 (0.15) (0.023) (0.016) (0.037) (Hamrick et al., 1992) Note: % P S – the per cent of polymorphic loci at the species level; A S – the number of alleles per locus at the species level; Н O – observed heterozygosity at the species level; Н E – expected heterozygosity at the species level; G ST (F ST ) – the parameter of subdivision. The values of standard mistakes are written in brackets Table 1. Polymorphism parameters in Aristolochia contorta compared to corresponding mean parameters for different plant groups In the populations of A. contorta the level of polymorphism is lower than in average for rare and endemic plants.

PopulationsHОHО HЕHЕ nЕnЕ AP 95, %P 99, % Romanovka Bol ’ shoi Kamen ’ Ussuriiskii region Oktiabr ’ skii region The average 0.09 (0.019) 0.09 (0.022) Table 2. Parameters of genetic variation in natural populations of Aristolochia contorta Parameters of genetic diversity in four populations studied were different. The highest level of the genetic variation was revealed in the population of the Oktiabr’skii region, whereas the lowest one was found in the Bol’shoi Kamen’ population. This fact may be explained by the most anthropogenic pressure on this territory. H O – observed heterozygosity, H E – the expected heterozygosity; n Е - the effective number of alleles per locus, A – the number of alleles per locus; P 95, P 99, %, polymorphism at the 95 and 99% criterion Bol ’ shoi Kamen ’ Oktiabr’skii region

Fig. 2. The distribution of allele frequencies of three polymorphic loci in Aristolochia contorta populations. Test for heterogeneity among all loci: χ2=109.58; df=15; р<0.01. Aat-1 (claret red and orange sectors of circuses correspond to allele frequencies 1 and 2), 6-Pgd-2 (green and yellow), Fe-1 (blue and yellow), Fe-2 (dark blue, cyan and white), Lap (orange, yellow, green and cyan). Additionaly some differences in allele set of Lap locus between all localities studied were found. Differences in allele frequencies among populations were statistically significant over all loci.

Table 3. F-statistics analysis of the Aristolochia contorta populations N e m=1.29 LociF IS F IT F ST Aat Pgd Lap Fe Fe Whole species F IS, inbreeding coefficient of individual respecting a population; F IT, inbreeding coefficient of individual respecting the whole species; F ST, the indicator of differentiation of populations. The Wright’s F-statistics analysis revealed a little excess of heterozygotes in the populations and, simultaneously, about 16% heterozygote deficiency for the whole species. It may be evidence of subdivision of the species into separate small reproductive groups. The differentiation index F ST varied from to and was 0.16 averaged for all loci. This value says that about 16.2% of the total variation is accounted for the inter-population covariation component. It is known, that population differentiation is connected to gene flow. This parameter was 1.29 among all populations. Obviously, gene flow between populations studied is not high enough for preventing the destructive consequences of genetic drift within small localities.

We estimated the genetic relationships among populations by Nei distances. Low differentiation of the localities evidences common gene pull among all populations and recent time of its separation. Populations’ isolation period, calculated by the formula of “molecular clock” is about 11.5 thousands years ago and corresponds to the beginning of Holocene when significant warming of climate occurred. We suppose that population of the Oktiabr’skii region, located on the southwest part of the Primorsky Krai, which avoided the glaciations in Quaternary period, was a refugium for A. contorta. After climate warming the species spread along Razdol’naya river and some it’s feeders, forming new populations. Obviously, environmental changing and possibly, economic development of this territory contribute to the weakening of the gene flow and increase of the population differ. D N =0.0229

Thus, this study showed a low level of genetic diversity and high level of genetic differentiation between Russian A. contorta populations. The data supposed that all the populations of A. contorta in Primorsky Krai have common gene pool. Today genetic drift is contributing the main input to the population genetic structure of the A. contorta. The enhancing of genetic drift is connected with reduction of reproductive and effective population sizes under increasing of anthropogenic pressure. The conclusion