[Species divergence in Iris series Lacteae (Iridaceae) in Russia and adjacent countries based on chloroplast DNA sequence data].

In Russia, the species of the genus Iris L., series Lacteae Doronkin, the taxonomic structure of which still remains controversial, are found in the south of Siberia and the Russian Far East, as well as in other regions of Asia. Sequence analysis of three chloroplast DNA regions (rps4, trnL­trnF, and trnS­trnG) shows that, in Russia and adjacent countries, there are two genetically and geographically isolated Lacteae species. I. oxypetala Bunge grows in the south of the Russian Far East, and I. lactea Pall. grows in Siberia, Mongolia, and Kazakhstan. Genetic differentiation between the populations of I. lactea is extremely low and statistically insignificant (the fixation index ΦST = 0.057, P > 0.05), pointing to the unity of the gene pool and the absence of other Lacteae species in this area.

[Phylogenetic relationships of the species of Oxytropis DC. subg. Oxytropis and Phacoxytropis (Fabaceae) from Asian Russia inferred from the nucleotide sequence analysis of the intergenic spacers of the chloroplast genome].

The nucleotide sequence analysis of trnH­psbA, trnL­trnF, and trnS­trnG intergenic spacer regions of chloroplast DNA performed in the representatives of the genus Oxytropis from Asian Russia provided clarification of the phylogenetic relationships of some species and sections in the subgenera Oxytropis and Phacoxytropis and in the genus Oxytropis as a whole. Only the section Mesogaea corresponds to the subgenus Phacoxytropis, while the section Janthina of the same subgenus groups together with the sections of the subgenus Oxytropis. The sections Chrysantha and Ortholoma of the subgenus Oxytropis are not only closely related to each other, but together with the section Mesogaea, they are grouped into the subgenus Phacoxytropis. It seems likely that the sections Chrysantha and Ortholoma should be assigned to the subgenus Phacoxytropis, and the section Janthina should be assigned to the subgenus Oxytropis. The molecular differences were identified between O. coerulea and O. mandshurica from the section Janthina that were indicative of considerable divergence of their chloroplast genomes and the species independence of the taxa. The species independence of O. czukotica belonging to the section Arctobia was also confirmed.

One or three species in Megadenia (Brassicaceae): insight from molecular studies.

Megadenia Maxim. is a small genus of the Brassicaceae endemic to East Asia with three disjunct areas of distribution: the eastern edge of the Qinghai-Tibetan Plateau, the Eastern Sayan Mountains in southern Siberia, and Chandalaz Ridge in the southern Sikhote-Alin Mountains. Although distinct species (M. pygmaea Maxim., M. bardunovii Popov, and M. speluncarum Vorob., Vorosch. and Gorovoj) have been described from each area, they have lately been reduced to synonymy with M. pygmaea due to high morphological similarity. Here, we present the first molecular study of Megadenia. Using the sequences of 11 noncoding regions from the cytoplasmic (chloroplast and mitochondrial) and nuclear genomes, we assessed divergence within the genus and explored the relationships between Megadenia and Biscutella L. Although M. bardunovii, M. speluncarum, and M. pygmaea were found to be indiscernible with regard to the nuclear and mitochondrial markers studied, our data on the plastid genome revealed their distinctness and a clear subdivision of the genus into three lineages matching the three described species. All of the phylogenetic analyses of the chloroplast DNA sequences provide strong support for the inclusion of Megadenia and Biscutella in the tribe Biscutelleae. A dating analysis shows that the genus Megadenia is of Miocene origin and diversification within the genus, which has led to the three extant lineages, most likely occurred during the Early-Middle Pleistocene, in agreement with the vicariance pattern. Given the present-day distribution, differences in habitat preferences and in some anatomical traits, and lack of a direct genealogical relationship, M. pygmaea, M. bardunovii, and M. speluncarum should be treated as distinct species or at least subspecies.

[Phylogenetic relations of Salix L. subg. Salix species (Salicaceae) according to sequencing data of intergenic spacers of chloroplasic genomes and ITS rDNA].

A phylogenetic analysis based on a comparison of nucleotide sequences of six regions from cpDNA and ITS rDNA (petN-psbM, trnD-trnT, trnC-petN, psaA-ycf3, petG-trnP, and rpoB-trnC) allowed for elucidating the relationship among species and sections belonging to the Salix subgenus and, more generally, to the Salix genus, as well as revealing the relations of the Chosenia genus. The definition of the subgenera of Pleuradenia (including the Urbanianea genus and the Chosenia genus), Salix (without the Triandrae section), Triandrae, and Longifoliae is essentially consistent with current classification schemes of the Salix genus. The previously defined genera of Chosenia and Toisusu (Urbanianea) are not only joined with the Salix genus but are also closely related between themselves. The Protitea subgenus only corresponds to the American species of the Humboldtianae section (S. humboldtiana, S. amygdaloides, S. gooddingii). The relationship of S. chaenomeloides, which is a nomenclatural type of this subgenus, as well as the relationship of the Wilsonia section, remains unresolved. The Humboldtianae section should be interpreted more narrowly, apparently, separating the Acmophillae and Tetraspermae sections from it. The monotypic American Floridanae section is related to the Salix, Salicaster, Tetraspermae, and Wilsonia.

[Phylogenetic relationships of Oxytropis chankaensis Jurtz. and Oxytropis oxyphylla (Pall.) DC. (Fabaceae) inferred from the data of sequencing of the ITS region of the nuclear ribosomal DNA operon and intergenic spacers of the chloroplast genome].

The autotetraploid Oxytropis chankaensis Jurtz. is the only representative of the section Baicalia (subgenus Oxytropis, Fabaceae) in Primorskii Krai, and its range is restricted to the sandy belt along the western coast of the Khanka Lake. The type species of the section Baicalia is O. oxyphylla (Pall.) DC., and O. chankaensis is regarded by some authors as its synonym. Analysis of the ITS sequences showed that O. chankaensis is phylogenetically closely related to the species O. oxyphylla and O. racemosa Turcz. from the section Baicalia and also to the species O. anertii Nakai., O. ciliata Turcz., and O. inschanica H. C. Fu & S. H. Cheng from the section Xerobia, but this analysis failed to resolve the phylogenetic relationships of the species within the genus Oxytropis. The analysis of the trnH-psbA, trnL-trnF, trnS-trnG, and petG-trnP regions of cpDNA revealed molecular differences between O. chankaensis and O. oxyphylla that are indicative of considerable divergence of their chloroplast genomes. The ancestral lineages of these species diverged nearly 2 million years ago, which confirms the taxonomic validity of O. chankaensis. Taking into account the ecological specificity of O. chankaensis and the present-day distribution range of O. oxyphylla, it can be assumed that they descended from one ancestral maternal lineage that gave rise to the species of the section Baicalia.

High chloroplast haplotype diversity in the endemic legume Oxytropis chankaensis may result from independent polyploidization events.

Oxytropis chankaensis Jurtz. (Fabaceae) is an endangered perennial tetraploid species endemic to the Khanka Lake coast. In Russia, O. chankaensis is distributed across a very restricted zone along the western shore of this lake. To characterise all known populations of this species, we assessed the genetic diversity of four noncoding regions of chloroplast DNA (cpDNA). Variable sites detected within the trnL-trnF, the petG-trnP, and the trnS-trnG regions allowed the identification of seven haplotypes. On the other hand, no variation was found in the trnH-psbA region. O. chankaensis exhibited an overall low level of nucleotide diversity (π = 0.00052) but a marked haplotype diversity (h = 0.718). A combination of three or four haplotypes was found in each population, and most of the cpDNA variation (above 90%) was distributed within populations. The level of genetic structure that we detected in O. chankaensis using maternal plastid DNA markers was much lower (G (ST) = 0.037) than the average that is estimated for angiosperms. We found no evidence for isolation by distance or for phylogeographic structuring in O. chankaensis. Our data suggest that autopolyploidy has arisen more than once in the evolutionary history of this species. Repetitive expansion and contraction during past and ongoing demographic events both seem to be involved in shaping the current genetic structure of O. chankaensis. This study provides valuable information for developing the most appropriate strategy for conserving this endemic species with a narrow habitat range.

[Genetic variation of Manchurian pheasant (Phasianus colchicus pallasi Rotshild, 1903) inferred from mitochondrial DNA control region sequences].

Sequence variation of the mitochondrial DNA control region was studied in Manchurian pheasants (Phasianus colchicus pallasi Rotshild, 1903) representing three geographic populations from the southern part of the Russian Far East. Extremely low population genetic differentiation (F(ST) = 0.0003) pointed to a very high gene exchange between the populations. Combination of such characters as high haplotype diversity (0.884 to 0.913), low nucleotide diversity (0.0016 to 0.0022), low R2 values (0.1235 to 0.1337), certain patterns of pairwise-difference distributions, and the absence of phylogenetic structure suggested that the phylogenetic history of Ph. C. pallasi included passing through a bottleneck with further expansion in the postglacial period. According to the data obtained, it was suggested that differentiation between the mitochondrial lineages started approximately 100 000 years ago.

[Independent species status of Iris vorobievii N.S. Pavlova, Iris mandshurica Maxim., and Iris humilis Georgi (Iridaceae): evidence from the nuclear and chloroplast genomes].

Nuclear (RAPD) and chloroplast (trnH-psbA, atpB-rbcL, rps4, trnL-trnF, and trnS-trnG) molecular DNA markers were used to study the populations of closely related species of the genus Iris L.: Iris vorobievii N.S. Pavlova, I. mandshurica Maxim., and I. humilis Georgi. An analysis of 243 RAPD fragments made it possible to calculate the main population parameters and to identify species-specific RAPD markers. Differences that discriminated the species were found in all but one (trnL-trnF) cpDNA region. The total length of the four variable cpDNA regions was 3640 bp, indels included. Their variation was low. A total of 22 variable sites (0.63%) were found; of these, 13 were parsimony informative and 9 were noninformative. Analysis of molecular variance (AMOVA) revealed significantly (P < 0.0001) high differentiation of both nuclear (F(ST) = 0.681) and chloroplast (F(ST) = 0.854) genomes for the species examined. Thus, a multigene approach confirmed the independent species status of I. vorobievii, I. mandshurica, and I. humilis and showed that I. vorobievii and I. mandshurica, but not I. humilis, are found in Primorye.

[Phylogenetic relationships of the Far Eastern Araliaceae inferred from ITS sequences of nuclear rDNA].

In eight species of the family Araliaceae, inhabiting the territory of the Russian Far East, the sequences of ITS regions of nuclear rDNA were determined. A comparison of these sequences enabled establishment of phylogenetic relationships between the Far Eastern and other members of the family. It was demonstrated that Aralia sensu populations from Primorye and Sakhalin were genetically different and, hereby, could be classified as interspecific taxa. Aralia continentalis along with A. cordata were attributed to the section Aralia sensu. Oplopanax elaus and O. horridus were found to be very close to each other, possibly being the subspecies of one species or relatively young species. Legitimacy of the isolation of two sections within the genus Eleutherococcus was confirmed.

[Analysis of genetic variation in rare endemic species Oxytropis chankaensis Jurtz. (Fabaceae) using RAPD markers]. / Analiz geneticheskoi izmenchivosti redkogo épidemichngo vida Oxytropis chankaensis Jurtz. (Fabaceae) na osnove RAPD-markerov.

The method of polymerase chain reaction with random primers (RAPD) was used to assess genetic variation and population differentiation in the rare endemic plant Oxytropis chankaensis Jurtz. (Fabaceae). DNA samples from plants of two isolated populations were compared at 133 loci detected by use of ten primers. Both populations examined were characterized by high polymorphism levels (P95 = 72.9%, A = 1.92 and P95 = 74.4%, A = 1.88, respectively). They were also statistically significantly different in the frequencies of most of the amplicons. For each of the plants, unique multilocus RAPD phenotype was established using 17 to 20 RAPD markers. Diagnostic markers were not revealed. The populations were poorly differentiated. On average, the between-population component accounted for about 8% of the variation, while 92% of the variation was detected within populations. High variation along with the low degree of differentiation characteristic of two most geographically remote populations of O. chankaensis can have several explanations, among which a polyploid origin of the species seems to be most important.

[Genetic variability and interrelationship of Siberian and Far Eastern larches from RAPD-analysis data]. / Geneticheskaia izmenchivost' i vzaimootnosheniia listvennits Sibiri i dal'nego vostoka po dannym RAPD-analiza.

Genetic diversity of larches from six geographically isolated regions, Tomsk, Irkutsk, Ulan-Ude (Siberia), and Blagoveshchensk, Khabarovsk, Yuzhno-Sakhalinsk (Far East) was examined by means of RAPD analysis. Tree DNA samples were compared using 457 RAPD loci (97% of which were polymorphic), identified with 17 primers of random sequences. In the samples examined, 32 to 49% of the genes were in heterozygous state, mean expected heterozygosity (Hexp) varied from 0.1373 to 0.1891, and the genetic distances (DN) for different sample pairs varied from 0.0361 to 0.1802. The main population parameters were determined for Larix sibirica Ledeb., L. gmelinni (Rupr.) Rupr., and L. kamtschatica (Rupr.) Carr. Analysis of the genetic relationships showed that L. kamtschatica was characterized by highest genetic differentiation from the other larches examined, while larches from Primorskii krai were genetically close to L. sibirica.

[RAPD- and allozyme analysis of genetic variability of Panax ginseng C.A. Meyer and P. quinquefolius L]. / RAPD- i allozimnyi geneticheskoi izmenchivosti Panax ginseng S. A. Meyer i P. quinquefolius L.

Inter- and intraspecific variation of two ginseng species Panax ginseng and P. quinquefolius was estimated by studying 159 RAPD and 39 allozyme loci. Parameters of polymorphism and genetic diversity were determined and a tree was constructed to characterize the differences between individual plants, samples, and species. Genetic variation in P. ginseng proved to be lower than in P. quinquefolius. Gene diversity in the total P. ginseng sample was comparable with the mean expected heterozygosity of herbaceous plants. This suggests that wild P. ginseng plants in various areas of the currently fragmented natural habitat and cultivated plants of different origin have retained a significant proportion of their gene pool. The mean heterozygosity calculated per polymorphic locus for the RAPD phenotypes is similar to that of the allozyme loci and may be helpful in estimating gene diversity in populations of rare and endangered plant species.

[Genetic relationships among Far Eastern species of the family Araliacea inferred by RAPD analysis]. / Izuchenie geneticheskikh sviazei mezhdu dal'nevostochnymi vidami semeistva Araliaceae metodom RAPD.

A molecular genetic study of Far Eastern species of the family Araliaceae by means of RAPD analysis was conducted. Using 21 primers we assessed variability at 595 loci. Based on matrices of genetic distances D, dendrograms of genetic relationships among eleven species of six genera of this family were constructed. Our results suggest that Acanthopanax sessiliflorus and Eleutherococcus senticosus belong to different genera, Aralia cordata and A. continentalis are different species, and A. elata and A. mandshurica probably cannot be regarded as distinct species. Genetic similarity of Far Eastern A. cordata and American A. hispida is shown.

Shikonin production by p-fluorophenylalanine resistant cells of Lithospermum erythrorhizon.

Studies were conducted with a BK-39 callus culture of Lithospermum erythrorhizon, which produced seven shikonin derivatives (acetylshikonin, propionylshikonin, isobutyrylshikonin, beta,beta-dimethylacrylshikonin, isovalerylshikonin, beta-hydroxyisovalerylshikonin and alpha-methyl-n-butyrylshikonin). A selection of cell aggregates of BK-39 culture on a medium containing p-fluorophenylalanine (PFP) yields a cell line possessing a higher resistance to the inhibitor than the initial culture. Selected BK-39F cultures produced almost the same profile of shikonin naphthoquinones as the initial culture. The shikonin derivative content of PFP-resistant culture was approximately two times higher than that of the control, reaching 12.6% of DW cell biomass.

[DNA from various tissues of the Far Eastern larch and its applicability for RAPD assay]. / DNK iz raznykh organov dal'nevostochnykh listvennits i ee prigodnost' dlia RAPD-analiza.

We studied applicability of RAPD assay for investigating genetic variability of far eastern larches. Techniques to isolate DNA from larch vegetative organs (needles, young shoots and stems) and megagametophytes were selected, and conditions of polymerase chain reaction were optimized. Fifty out of 84 random primers proved efficient, and 22 and 12 primers initiated weak and no synthesis, respectively. The tested primers worked efficiently on the DNA isolated from both diploid and haploid tissues.

[Genetic variability of Iris setosa]. / Issledovanie genetichskoi izmenchivosti Iris setosa.

Genetic variability of Iris setosa Pall. ex Link. was studied by the RAPD method. Plants from three different habitats were compared by 135 loci revealed with eight arbitrary primers. The three plant accessions all exhibited a high level of polymorphism, and each was characterized by different frequencies of polymorphic fragments, which probably reflected the geographic isolation of the analyzed populations. The average level of polymorphism detected was 35%.

[Use of RAPD-PCR-analysis of cellular DNA for the evaluation of genetic polymorphism and subspecies diagnostics of the Far Eastern leopard Panthera pardus orientalis]. / Ispol'zovanie PP-PTsR-analiza kletochnoi DNK dlia otsenki geneticheskogo polimorfizma i podvidovoi diagnostiki dal'nevostochnogo leoparda Panthera pardus orientalis.

Genetic polymorphism in the Far-Eastern leopard subspecies Panthera pardus orientalis was examined by RAPD-PCR analysis of total cellular DNA. Primers diagnostic for the leopard subspecies were detected. These primers can be used for differentiating wild-living animals from those kept in captivity and distinguishing different breeding lines. Genetic diversity was shown to be lower in animals from the zoo (D = 0.058) and higher in a natural population (D = 0.137). Two lines of the Persian subspecies were genetically closest (D = 0.042), and the Persian and Far-Eastern subspecies represented by wild animals, genetically most remote (D = 0.274). The level of genetic divergence of the Persian subspecies and Far-Eastern subspecies (zoo animals) was lower (D = 0.108) than between representatives of the Persian subspecies from the captive and natural population (D = 0.220). On the basis of analysis of relatedness, performed using NTSYS and PHYLIP software packages, the possibility cannot be excluded that some P. p. orientalis animals from the zoo have a hybrid origin and may be assigned to another Far-Eastern subspecies.