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Polymorphism and genetic structure of populations Orchis militaris and O. simia, in the Crimea and in the Caucasus, with the use of allozyme analysis of 8 genetic loci (PGI, NADHD, SKDH, GDH, PGM, DIA, ADH, IDH) is studied. The percentage of polymorphic loci in different populations of O. militaris ranged from 12.5 % to 75 %. The greatest value is characteristic for the populations from the Krasnodar region. The percentage of polymorphic loci in different populations of O. simia varied from 62 % to 75 %. Two loci (ADH and NADHD) were identified, which have the taxon-specific alleles, in which O. militaris and O. simia individuals differ significantly. The polymorphism of certain alleles at the loci IDH and PGI in populations of O. militaris and the IDH and PGM loci in populations of O. simia, apparently, is associated with interspecific introgressive hybridization, as revealed only in the area of joint growth of individuals of two species. The genetic differentiation index between 4 populations of O. militaris (Fst) averaged 0,075, which according to the Wright scale of assessment (Hedrick, 2003) is mean value. Genetic differentiation between populations is expressed to a high degree by the allelic composition of the ADH locus (Fst=0.241). At loci GDH Fst comprised 0.097, that it corresponds to average value. The genetic differentiation index between the 3 populations of O. simia (Fst) was 0.071, which refers to the mean values. The analysis of genetic distances according to Nei (1978) testifies to insignificant geographical differentiation of O. militaris populations on the most part of an area and to isolation of the populations growing in the Caucasus.
The results of the study of the polymorphism of representatives of the genus Cypripedium from allozyme analysis and the trnL region of chloroplast DNA, as well as the 454 (pyrosequencing) study of intra-genomic polymorphism of ITS1 are presented. Allozyme analysis was performed using 10 gene loci in individuals from the populations of Transbaikalia, Central and Western Siberia, the Urals and the European part of Russia, which supplemented previously obtained data on populations from the south of the Far East. It has been confirmed that the allelic compositions of the 6-PGD, NADHD, GDH, ADH, GOT-1 and PGI loci are diagnostic for C. calceolus and C. macranthon species, C. × ventricosum are C. calceolus × C. macranthon hybrids, C. shanxiense specimens are homozygous for all 10 loci studied, and the sign of monomorphy is diagnostic for a given taxon. It was shown that the allelic composition of PGI and SKDH loci in C. calceolus varies from western and eastern parts of the range. Alleles, absent from C. calceolus from the western part of the range, were encountered in representatives of the species in the eastern part with a high frequency. Thus, allele No. 6 in the PGI locus and allele No. 4 in the SKDH locus was found in C. calceolus at a frequency of 28-41% and 55-68%, respectively, in the south of the Far East, and at a frequency of 0-15% and 17-62% in Transbaikalia, and in C. shanxiense they were in a homozygous state. These alleles were not found in populations of C. calceolus from Central and Western Siberia, from the Urals, from the European part of Russia. Thus, the study confirmed the view that the special allelic structures of PGI and SKDH loci in C. calceolus populations are found only in the zone of possible hybridization with C. shanxiense (in places of joint growth of two species and the adjacent territory). On the basis of allele frequencies, the genetic distances of DA by Nei et all. (1983) were calculated and UPGMA dendrogram was constructed. It is shown that individuals of C. calceolus, found on the territory of Russia, form two groups, the border between them passes through the Transbaikal. Morphological analysis revealed that a large number of individuals with atypical traits (a flower with a brown lip) and features of reproductive biology (facultative self-pollination) are present in the populations of C. calceolus growing in the south of the Primorsky, Khabarovsk and Zabaikalsk territories. There is genetic instability in the formation of pollen and seeds and, as a consequence, a decrease in seed production. Identified atypical morphological and molecular genetic (based on allozyme analysis) signs in C. calceolus, which were observed only in individuals in the zone of joint growth with C. shanxiense, were in good agreement with the assumption of interspecific introgressive hybridization between these taxa. It was suggested that there is an interspecies introgressively hybrid complex in the zone of overlapping of the ranges of C. calceolus and C. shanxiense in the Transbaikal, Khabarovsk and Primorye regions, where hybrids (C. calceolus × C. shanxiense) predominate, and the number of individuals of parental species is comparatively small. One of the parental species - C. shanxiense, belongs to the rarest species of the genus, is characterized by obligate autogamy, which provides biological isolation of the given taxon from the closely related species C. calceolus. Representatives of the second of the parent species, C.calceolus, are perhaps even more rare in this area, and the plants morphologically similar to C. calceolus are in fact hybrids of C. calceolus x C. shanxiense. To confirm this hypothesis, a study of intragenomic polymorphism was carried out using the technique of 454 sites of 5.8S ribosomal (ITS1) DNA and chloroplast DNA of the trnL-F region. Analysis of the ITS1 site in 16 C. calceolus samples showed that some of them have an intra-genomic polymorphism at position 320 (the position is hereinafter denoted according to a similar reference in the reference sequence of C. calceolus from the genebank (sample of Europe)). In three samples, sequences similar to the one in the same sample prevailed in one sample, sequences with replacement of C by T prevailed. Analysis of the ITS1 region of 7 samples of C. shanxiense (4 samples from Transbaikalia, 2 from Sakhalin, 1 from Primorye) showed that in positions 268 and 276 There are replacements of G by T, and at position 320-C on T. As it turned out, replacements in positions 268 and 276 are specific for a given taxon, and intra-genomic polymorphism takes place according to them. So, samples from Transbaikalia differed from the reference in positions 268 and 276 in 50-79% of readings, from Primorye - in 85-87%, and from Sakhalin 57-73%. Analysis of the ITS1 region of 5 samples of C. macranthon showed that they differed from the reference at positions 269, 320 and 352, where there are substitutions of C to T. In positions 269 and 352 they differ also from C. calceolus and C. shanxiense. Analysis of the ITS1 region of 11 samples of C. × ventricosum showed that there is polymorphism at positions 269 and 352, which distinguish the parent species of C. calceolus and C. macranthon. The number of readings with replacements in positions 269 and 352 ranged from 30 to 50%. According to the results of the study, almost all C. × ventricosum samples are hybrids of the first generation between C. calceolus and C. macranthon. These data are completely consistent with the results of allozyme analysis. No hybrids between C. macranthon and C. shanxiense were found. In two of the 11 samples of C. × ventricosum studied, the studied region of chloroplast DNA resembled C. calceolus from the Transbaikalian Territory and from Primorye, in all others it was identical to that of C. macranthon. Thus, the individuals of this hybrid taxon can be formed with participation of both C. macranthon and C. calceolus as female plants. If the studied samples of C. calceolus did not differ in the ITS1 region, then they were divided into 2 groups according to the chloroplast DNA. The nucleotide sequence of the region of chloroplast DNA was identical in all the C. shanxiense and C. calceolus specimens studied from the Transbaikalian Territory and from Primorye. However, when compared with the samples of C. calceolus collected in the Transbaikalian part of Buryatia and to the west (Siberia, the Urals and the European part of Russia), there were significant differences in the presence of insertions / deletions at positions 457 and 516. The probable boundary between the groups of C. calceolus occurs in Trans-Baikal Territory. These data are in good agreement with the results of allozyme analysis. 142 variants of ITS1 sequences were analyzed, which were found in the genome of the analyzed samples with a share of at least 5% and had a level of site variability of approximately 80%. The analysis divided the options into two large clusters and into 33 groups. It was noted that the groups include identical ITS1 variants found in samples of different taxa. Variants of C. calceolus came in 3 groups, only together with samples of C. × ventricosum and C. shanxiense. The variants of C. shanxiense were divided into 9 groups (6 of which are specific only for a given taxon). C. macranthon variants are found in 10 groups, in almost all of them there are C. × ventricosum samples. Variants of C. × ventricosum were divided into 23 groups, as a rule, they contain either samples of C. calceolus or C. macranthon. No group combining variants from both C. calceolus and C. macranthon, or both from C. shanxiense and from C. macranthon, was identified. The data obtained indicate that there is apparently no interspecific hybridization between C. shanxiense and C. macranthon in sympatric populations. From the obtained results it follows that on the territory of Russia there are two different genetic lines of C. calceolus, which differ in the chloroplast DNA. The region of chloroplast DNA in one of the lines of C. calceolus and C. shanxiense was identical, whereas the ITS1 portion of DNA in the representatives of the two taxa had significant differences. It was noted that the individuals of this line of C. calceolus grew only in the zone of the locations of C. shanxiense. The hybridization zone between C. calceolus and C. shanxiense, determined on the basis of the allelic composition of PGI and SKDH loci (by differentiating C. shanxiense alleles in C. calceolus) appears to be wider, It extends not only to the territory of joint growth of two taxa, but also to the adjacent areas. This may be due to the fact that there is a flow of genes that is carried through the transfer of pollen over long distances, compared with the dispersion of seeds. However, the exchange of genetic material between taxa is very rare. As it turned out, not all the hypotheses expressed on the basis of the data of the allozyme analysis were confirmed after the investigation of the intragenomic polymorphism of the ITS1 region of the DNA. For example, the difference in the allelic composition of the two allozyme loci PGI and SKDH in C. calceolus from the eastern and western parts of the range could be explained by the flow of genes from C. shanxiense, which in sympatric populations can also occur in real time. However, in the study of intragenomic polymorphism of the ITS1 region of DNA in C. calceolus, the replacement of specific C. shanxiense was very rare (no more than 1.5% of all readings). This clearly indicates that the analyzed samples of C. calceolus (including two atypical specimens with a brown lip) are not hybrids of the first generation. Whereas in the genome of C. shanxiense, which, according to the allozyme analysis, is monomorphic in all 10 gene loci, which does not make it possible to attribute it to hybrids, there were from 13% to 50% identical C. calceolus ITS1 sequences. According to these data, C. shanxiense, could be attributed to interspecific hybrids, with the flow of genes from C. calceolus. However, the lack of polymorphism of allozyme loci in C. shanxiense suggests that all the samples studied (even those with a fraction of the sequence specific for C. calceolus ITS1 were 50%) are also not hybrids of the first generation. Taking into account both the data of allozyme analysis and the results of the investigation of intragenomic polymorphism, it can be concluded that at present there are no facts confirming the presence of hybridization between C. shanxiense and C. calceolus in sympatric populations. Based on the results of the study, it is possible to suggest the hybridogenic origin of both C. shanxiense and the "eastern" race of C. calceolus, spread from the Trans-Baikal Territory to Primorye. Perhaps this race of C. calceolus, characterized by pronounced phenotypic and genotypic features, should be considered as an independent morphologically weakly expressed latent taxon of a certain taxonomic rank.
Проанализирована жизнеспособность семян и семенного потомства Orchis purpurea ssp. purpurea с использованием методики проращивания семян в культуре in vitro. Для посева использовали незрелые семена из двух местонахождений полуострова Крым. Проведено цитоэмбриологическое исследование семян на момент посева, непроросших семян через год после посева и протокормов, остановившихся в развитии сразу после выхода из семенной кожуры. Показано, что в 2016 году процент завязывания плодов варьировал от 9 % (лес, с. Скалистое) до 39 % (поляна на склоне, с. Перевальное), т.е. он был существенно выше у особей, растущих на открытом участке. Семенное потомство было получено только от 14 особей (из 43 использованных в работе), из них от 10 это были лишь единичные сеянцы. Данные подтвердили точку зрения о низкой реальной семенной продуктивности (РСП) у изученного вида. Цитоэмбриологическое исследование установило, что РСП снижается из-за недостаточности опыления, летальных аномалий развития суспензора и покровов семени и из-за массово проявившейся неспособности проростков к формированию апекса побега. The viability of seeds and seed posterity of O. purpurea ssp. purpurea are analyzed with the use of seed germination in the culture in vitro method. For the sowing used unripe seeds of two locations of the Crimea. Cytoembryological study of seeds, at the moment of sowing, the ungerminated seeds in a year after sowing and protocorms, which were stopped in the development immediately after output from the seed coat is carried out. The predominantly seed propagation is characteristic for O. purpurea, but the percentage of fruit sets, as a rule, low. In the present investigation in 2016 the percentage of fruit sets varied from 9 % (forest) to 39 % (clearing on slope), i.e., it was substantially above in individuals, who grow in the open places. In the studied locations per year of a study it was somewhat higher than according to the information from the literary sources. Seed posterity was obtained only from seeds of 14 individuals, from 43 of plants used in investigation. From 10 plants were received only single seedlings. Thus, the results of the carried out experiments for seed germination confirmed the point of view about the low real seed productivity in the studied species. Cytoembryological study showed that the large part of the seeds, used for the sowing, had an embryo. Only in the 2 individuals (from 43) were discovered both seeds with the small embryo and ovules with the embryo sacs. Thus, it is possible to conclude that a few ovules could not be fertilized. However, the facts of the insufficiency of pollination among the studied fruits were single. The seeds, collected in the different locations, were on the different stages of development at the moment of sowing. In the embryo cells of some individuals noted the beginning of globules of protein formation. The accumulation of storage nutrients in embryo testifies about the begun process of ripening seeds and about shaping of the mechanism of braking their germination. Such seeds did not germinate in the experiment on nutrient medium. Apparently, the stage of their development was not optimum. Investigation of formed seed structures of individuals, when the seedlings were not obtained entirely, or only in the single number, was noted that multiple internal integument takes place in micropilar part of seeds. Thus, a micropyle was formed not only external integument and funiculus as this must be in normal, but also internal integument. This is evident disturbance of the process of the seed coat development. As a rule, in such seeds were observed the anomalies of the structure of suspensor. According norm formed suspensor in the Orchis is filamentary, consists of 7-9 the uniform vacuolized, elongated along the apical-basal axis mononuclear cells. It leaves through micropyle and takes root in the tissue of the placenta of ovary (Nikiticheva,1997; Lagutova, Chebotar, 1992). For used in the present investigation seeds of majority plants, suspensor did not exceed the limits of the seed coat It was located in the micropyle, as a rule, exceed the board of the internal integument, in the form winding structure, which consists of one cell row. The cells of suspensor, which adjoin to the body of embryo, as a rule, were not elongated along the apical-basal axis as this would be must according to norm. Obtained data testify about the disturbance of morpho-physiological correlations into the development both the seed covers and the embryo. Apparently, these anomalies relate to the lethal. The mass number of plantlets was obtained only for those maternal plants, whose seeds did not have anomalies in the structure of suspensor and seed covers. In a few cases into micropylar part of the seed were noted the formations, formed from the cells of internal integument, which had a similarity to the integumental embryos, described in Spiranthes cernua (Swamy, 1948). However, in the present experiments with the seed germination additional embryo did not discover. It is likely, either additional embryos were nonviable and dyed at the early stages of development, or structures in massive internal integument had only formal resemblance to the embryoids. Thus, massive internal integument, uncommonly long time being been preserved into micropylar part of the seed were not capable to the formation of additional embryos even under the conditions of the cultivation in vitro. It took notice of the uncommon fact of the mass loss of seeds at the initial stage of their germination. Cytoembryological study showed that during germination cells of embryo increased in size without their division, the forming structure lacked zone of small cells and the shoot apex. On the basis the results of a cytoembryological study is made the conclusion that in O. purpurea ssp. purpurea from 2 locations of Crimea occurs low real seed productivity because of the insufficiency of pollination, lethal anomalies of the development of the seed covers and suspensor and because of the mass appeared incapacity of seedlings to form of the shoot apex. The revealed anomalies of the development of seeds and seedlingss were encountered only in O. purpurea ssp. purpurea from the studied locations of the Crimea, they were not discovered in the conducted earlier investigation O. purpurea ssp. caucasica from the Caucasus.
The study of relationships between symbiotic fungi and germinating seeds of the Orchidaceae showed that the effect of a fungus on germination depends on the presence of seed dormancy and its pattern. The specificity of symbiosis varies widely. The development of symbiotic sprouts in situand in vitro differs only in the rate, whereas the morphogenesis of asymbiotic sprouts is characterized by several anomalies.
A cytoembryological study of the seed formation in Orchis purpurea Huds. ssp. purpurea from two populations of Krasnodar region, which are characterized by low real seed productivity, is carried out. It is established that the seeds become nonviable up to the moment of dissemination because of the lethal anomalies in embryo and suspensor development, that are connected with the manifestation of genetic instability in the division and differentiation of cells during embryogenesis.
The date of the grown in the culture in vitro number dynamics of the Dactylorhiza maculata s.l. plants after their transplantation into natural conditions (Moscow reg.), and also with respect to a change in their morphometric parameters for 1 – 5 years of growth on the experimental sites in the different phytocoenosis are generalized. It is shown that the number of experimental plants is reduced during first three years, and for the fourth or fifth years its stabilization is observed. The majorities of the plants of the first after transplantation year, as a rule, had 2 green sheets, and the large part of plants the second and subsequent years only 1 green sheet. Their remaining morphometric parameters (length, the width of sheet, the number of veins) gradually increased from year to year. The experimental plants through are 2nd or 3 years after transplantation although have the similar morphometric parameters, can be distinguished by type the shoot growth (monopodial or sympodial). Possibly, the prolonged development of experimental plants is connected with the delay of their seasonal development in connection with the less favorable conditions of the planting site.
The study is carried out of seeds at different stages of their development in an individual of Dactylorhiza fuchsii (Druce) Soó, characterized by a high share of nonviable seeds by the moment of dissemination. It is shown that the embryo is present in the majority of the seeds at the early stage of their development. In most cases, however, structural changes are observed in embryos: irregular cell divisions during the embryo body formation and a chaotic arrangement of partitions during their division, change in size and shape of the suspensor cells, delay of the divisions of certain cells, development of the polyploidy nuclei which are the indications of genetic disturbances in the normal way of embryonic growth. In the process of seeds ripening, such underdeveloped and anomalous embryos gradually degenerate, therefore up to the moment of dissemination the part of the seeds in the studied plant become nonviable.
During investigation of wild Cypripedium populations in the Far East and the Khabarovsk Region (June 2002, 2005) the cjntact autogamy was found in all investigated plants of C. shanxiense and a few ones of C. calceolus. Morphological characterictic features of a flower in autogamous plant are following: 1) the anthers and the stigma disposed at a short distance 9less then 1 mm); 2) unite drop of amorphous sticky pollen mass formed after the anthers opening; 3) the pollen mass can run from anther surface to stigma. 2-16 % autogamous plants were found in each of 5 investigated populations of C. calceolus. Usually only one anther takes part in self – pollination. The share of autogamous flower was 25 – 66 %, very seldom 100 %. A facultative autogamy is concluded to take place in C. calceolus in the Far East.
The article provides data on the fruit set, heterospermy and quality of seeds in Orchis purpurea subsp. caucasica. The species is rare and listed in the Red Data Book of Russian Federation (2008). It propagates mainly by seed. It is shown that in the studied localities in Novorossiysk and Abinsk districts of Krasnodar Territory in 2009 – 2014 the average share of fruit set was very low (1–4%). Seeds of O. purpurea from the fruits of different plants differed in viability, color of seed coat and embryo, as well as the degree of uniformity of their dimensions (length, width) and shape. The share of nonviable seeds (without embryo) in the fruits developed after natural pollination was 50% on the average. It is shown that the share of nonviable seeds in the fruits developed after geitonogamy was substantially higher than in those after xenogamy. It is suggested that the low share of fruit set and the low seed quality can be related to both non-optimal conditions in the localities (extreme sites at the range limit) and non-optimal weather conditions for pollination and development the plants in the years of the study. The high share of abnormalities during the seed development may probably indicate the hybrid origin of parent plants, or the hybrid nature of progeny as a result of pollination by another species. Furthermore, it is possible that the high share of nonviable seeds may result from inbreeding connected with a small number of individuals in the studied populations. It is assumed that in the studies areas of the North-Western Caucasus an interspecific introgressive hybrid complex of several Orchis species has been probably formed. This may explain a high polymorphism and a low actual seed productivity of the Orchis purpurea.
25.07.2016 г. С использованием аллозимного анализа изучены полиморфизм и генетическая структура популя-ций D. incarnata и D. ochroleuca в местах их совместного произрастания в России и Беларуси. Пока-зано, что особи D. ochroleuca из популяций с Урала и Сибири, удаленных от основной части ареала, не отличаются от особей из основной части ареала (Беларусь) по аллельному составу восьми генных локу-сов. Выявлено, что D. ochroleuca и D. incarnata дифференцированы по разным аллелям локуса GDH. Тем самым был установлен генный маркер, по которому эти близкие таксоны различаются. Кроме локуса GDH особи D. ochroleuca и D. incarnata в зоне их совместного произрастания различаются по аллель-ной структуре PGI и NADHD. Несмотря на то что у D. incarnata с Урала и из Сибири оба локуса были полиморфными, а из Беларуси-один из них (PGI), все особи D. ochroleuca, произрастающие сов-местно с полиморфными особями D. incarnata, оказались гомозиготными по одинаковым аллелям. Сделан вывод о генетической обособленности и существовании механизма изоляции D. ochroleuca от D. incarnata даже при их совместном произрастании. Обнаружено, что локус GDH у D. incarnata полиморфен только в популяциях, которые произрастают совместно с D. ochroleuca, за исключени-ем единичных примеров. Было сделано заключение, что изменчивость локуса GDH у D. incarnata связана с гибридизацией с D. ochroleuca. Ключевые слова: Dactylorhiza incarnata, D. ochroleuca, аллозимный анализ, межвидовые интрогрессив-но гибридные комплексы, генетический полиморфизм.
28.03.2016 г. Представлены результаты изучения полиморфизма и генетической структуры популяций Dacty-lorhiza salina и D. incarnata, произрастающих в Забайкальском крае и Бурятии, по данным аллозим-ного анализа восьми генных локусов (PGI, NADHD, SKDH, GDH, PGM, DIA, ADH, IDH). Установле-на специфичность аллельной структуры локусов SKDH, PGM и IDH для D. salina и D. incarnata, по которым виды достоверно отличаются друг от друга. Показано, что в Забайкалье сформировались межвидовые интрогрессивно гибридные комплексы, имеющие разную генетическую структуру. В местах массового произрастания D. incarnata встречаются единичные растения D. salina, межвидо-вые гибриды первого и последующих поколений. В местах массового произрастания D. salina обна-ружены только гибриды, не являющиеся гибридами первого поколения. Они были гетерозиготны-ми не по трем локусам с дифференцирующими аллелями обоих родителей-SKDH, PGM и IDH, а лишь по одному из них. Степень генетической дифференциации между пятью популяциями D. sa-lina в среднем составляла 7.5%, а для D. incarnata-7.1%, что в соответствии со шкалой оценки Райта относится к средним значениям. Среднее значение F ST для всех изученных популяций двух близких видов рода Dactylorhiza составило 0.478, что свидетельствует об очень высокой степени генетической дифференциации между D. salina и D. incarnata, произрастающих в Забайкалье. Наиболее сильно виды различаются по аллельной структуре локусов SKDH, PGM и IDH (F ST равнялся 0.705, 0.976 и 0.762, соответственно). Дисперсионный анализ данных (AMOVA) показал, что между популяциями D. salina и D. incarnata, в зоне перекрывания их ареалов в Забайкальском крае и Бурятии, имеются существенные различия, и в варьировании частот аллелей восьми локусов (71%, d.f. = 9), и в измен-чивости генотипов (61%, d.f. = 9). Несмотря на то что между D. salina и D. incarnata существует явный поток генов в результате межвидовой гибридизации, генетическая дифференциация популяций этих близких видов сохраняется на высоком уровне. Ключевые слова: Dactylorhiza salina, D. incarnata, аллозимный анализ, межвидовые интрогрессивно гибридные комплексы, генетический полиморфизм.
The results of studying the polymorphism and genetic structure of populations of D. salina and D. incarnata growing in Zabaykalsky krai and Buryatia are represented according to the data of allozyme analysis of eight genetic loci (PGI, NADHD, SKDH, GDH, PGM, DIA, ADH, and IDH). The specificity of the allelic structure of loci SKDH, PGM, and IDH is established, for which D. salina and D. incarnata reliably differ from each other. It is shown that interspecies introgressive hybrid complexes with different genetic structures were formed in Transbaikalia. Places of mass growth of D. incarnata were observed to have single plants of D. salina, the interspecies hybrids of the first and subsequent generations. Places of mass growth of D. salina were observed to contain only the hybrids that are not hybrids of the first generation. They were heterozygous not for three loci with differentiating alleles of both parents, SKDH, PGM, and IDH, but for only one of them. The degree of genetic differentiation among five populations of D. salina was on average 7.5% and that of D. incarnata was 7.1%, which in accordance with Wright’s estimation relates to mean values. The average value of FST for all studied populations of the two related species of the genus Dactylorhiza was 0.478, indicating a very high degree of genetic differentiation between D. salina and D. incarnata growing in Transbaikalia. The greatest differences between the species are for the allelic structure of loci SKDH, PGM, and IDH (FST was equal to 0.705, 0.976, and 0.762, respectively). Analysis of molecular variance (AMOVA) showed that populations of D. salina and D. incarnata in the zone where their ranges in Zabaykalsky krai and Buryatya overlap have essential differences both for the variation of alleles frequencies of eight loci (71%, d.f. = 9) and for the variability of genotypes (61%, d.f. = 9). Despite the fact that D. salina and D. incarnata explicitly share a gene flow as a result of interspecies hybridization, the genetic differentiation of populations of these related species remains at a high level.
Система скрещиваний в изученных популяциях Cypripedium на юге Хабаровского и Приморского краев шире, по сравнению с таковой в популяциях, произрастающих в других частях ареала. Во-первых, за счет увеличения числа видов, участвующих в гибридизации. На Даль-нем Востоке совместно произрастают четыре вида: C. ventricosum, C. calceolus, C. macranthon и C. shanxiense. Во-вторых, увеличивается доля семенного потомства, формирующегося за счет самоопыления. Тем самым, усиливается роль инбридинга, и, следовательно, увеличивается вероятность гомозиготности. Все это определяет особенную генетическую структуру популяций Cypripedium на юге Хабаровского и При-морского краев и большой полиморфизм, наблюдаемый именно в дан-ной части ареала.
We carried out an allozyme analysis to investigate polymorphism and genetic structure of the populations of D. incarnata and D. ochroleuca in regions of their joint growth in Russia and Belarus. We found that D. ochroleuca individuals in the populations of the Urals and Siberia, which are distant fragments from the main range of the species, do not differ significantly from individuals within the main part of the area (Belarus) on the basis of the allelic composition of eight gene loci. We revealed that D. ochroleuca and D. incarnata are differentiated by different alleles of the GDH locus. Thus, we established a genetic marker suitable to distinguish these closely related taxa. In addition to the GDH locus, D. ochroleuca and D. incarnata in the places of their joint growth, differ in the allelic structure of the PGI and NADHD loci. D. incarnata from the Urals and Siberia were polymorphic for both loci, and individuals from Belarus were polymorphic for one locus (PGI). In contrast, all D. ochroleuca individuals growing in sympatric populations with polymorphic D. incarnata were homozygous for the same alleles. Thus, comparison of the genetic structure of D. ochroleuca and D. incarnata points to the existence of a genetic isolation and a functioning isolation mechanism even under conditions of their joint growth. We found that the GDH locus in D. incarnata is polymorphic only in populations which grow together with D. ochroleuca, with exception a few examples. Thus, we conclude that variability of the GDH locus in D. incarnata is associated with hybridization with D. ochroleuca.