Annotation of the results obtained in 2021
As a result of the work performed during the reporting period, we have a high-quality herbarium and living materials, and samples for DNA extraction harvested at the population level and from different areas of the ranges of the studied taxa. To identify cryptic species and hybrid taxa, materials were primarily harvested in the extreme areas of the range and from mixed populations. During the reporting period and previous stages of the project on the genus Eranthis, the material was collected from 198 populations for all known species within their entire range; new habitats were found. Analysis of the literature on the genus Eranthis showed lack of data on the pubescence of peduncles and insufficient description of important characteristics of flower parts (petals). Previously, European representatives of the genus were considered to have bare peduncles, and rare pubescence, and their absence was indicated for Asian species. A detailed study of natural populations showed the pubescence of peduncles in all species of the genus. It was found that each species has a unique combination of characteristics, and all species have simple unicellular hairs. In addition to hair types, such characteristics as pubescence density, hair length and width (basal and apical) were explored. It was shown that despite the morphological similarity of plants from local populations over a wide discontinuous range of the species, the nature of the pubescence of peduncles in combination with some other morphological characteristics (statistically analyzed), biochemical composition, karyotype features and sample topology in phylogenetic trees indicates the presence of cryptic species and hybrid taxa. A phylogeny has been built for all populations; however, primarily important issues are nomenclature, identification of morphological differences within controversial populations and species with an uncertain taxonomic status, and obtaining biochemical and cytogenetic data, which are partially resolved. Within the group of European species, 2 large clades with 5 subclades are distinguished, 1 confirmed cryptic species has been identified (requires description). The ranges of Eranthis hyemalis and E. cilicica do not overlap, yet the presence of hybrid taxa should be confirmed, since cases, when parental species are lacking and hybrid species with a vegetative reproduction path have existed for a long time, are known for the Eudicot group (Grant, 1985). Comprehensive analysis of the Asian group of white-flowered Eranthis species indicated a greater number of extant species than previously thought. The recently described species Eranthis tanhoensis was identified through an integrative approach (Erst et al., 2020). Within the Asian group, 2 large clades with 8 subclades are distinguished. Additional materials collected in 2020–2022 and the materials provided by collaborators showed several new cryptic species identified within the Asian group. However, several blocks of work and analysis of all varieties should be completed to perform a more detailed study. As a result, the taxonomic diversity of the Ranunculaceae fam. was revised for a number of hard-to-reach regions of Asia. The findings were published in 3 scientific papers in PhytoKeys (Q2, IF – 1.635) and Phytotaxa (Q3, IF – 1.171) The study of the origin and evolution of taxa, their relationship with the phylogenetic trees is one of the priorities of modern biology. In recent decades, invaluable advances made included accumulation of a huge array of phylogenetic information, and reconstruction of the main branches in the Ranunculaceae group. Despite this, many parts of the tree have not yet been elaborated. This is primarily due to the lack of a complete set of species among researchers, hybridization processes, reticulate evolution, and adaptive radiation. Many examples of that kind are encountered in the fam. Ranunculaceae, in particular, unresolved issues arise in taxa undergone explosive adaptive radiation associated with rapid speciation over short periods of time. Complex problems of fundamental biology can be solved using next generation sequencing (NGS) genomic methods. In addition, these methods can be used to identify rapidly evolving loci and promising molecular markers that will be useful for future comprehensive studies. The results will provide abundant information for further species identification, phylogenetic, and phylogeographic studies on Thalictrum. The plastid genome sequences have been utilized successfully for the phylogenetic studies on angiosperms (Li et al., 2019). Our phylogenetic trees based on whole complete plastid genome sequences, 116 IGS regions, and 114 gene sequences revealed that Thalictrum contains two major clades that is consistent with previous studies (Morales-Briones et al., 2019). However, the relationships along the backbone of the clades are not well supported in their studies. None of the sections traditionally circumscribed for this genus is monophyletic. It is necessary to apply more samplings and find more efficient molecular markers for Thalictrum. Our phylogenetic trees indicated that 116 IGS regions had stronger support than 114 gene sequences. Additionally, the rpl16 intron—that was used by Soza et al. (2012) with high sequence divergence in the studies—showed also strong support in clades II here. While the coding regions of rbcL employed by Wang et al. (2018) showed lower supports within clades II in our analysis. The non-coding regions (introns and spacers) are more variable molecular markers. For the ML tree of rpl32-trnLUAG used by Wang et al. (2018), the outgroups are embedded in Thalictrum probably because the matrix of rpl32-trnLUAG contained lots of indels. The rpl32 gene is often transfers to the nucleus (Park et al., 2015) that make the ndhF-rpl32, rpl32-trnLUAG, and rpl32 regions not reliable to be markers for phylogeny in Thalictrum. Our phylogenomic analyses based on whole complete plastid genome sequences, 116 IGS regions and 114 gene sequences were all supported the monophyly of Thalictrum and two major clades within this genus. Furthermore, among 79 plastome-derived protein-coding genes (CDSs), 15 genes were identified as fast evolving genes, which were all proved to be under positive selection but showed different bias in their codon usage frequencies. Overall, our results demonstrate the ability of plastid phylogenomics to improve phylogenetic resolution, and will expand the understanding of plastid gene evolution in Thalictrum. The study results were published in Frontiers in Plant Science (Q1, IF – 5.75). Cytogenetic analysis for a number of fam. Ranunulaceae species was conducted in the following directions: comparative analysis of chromosome sets (karyotypes), localization of 45S и 5S ribosomal DNA (rDNA) on chromosomes by fluorescence in situ hybridization (FISH) with oligonucleotide DNA-probes, determination of the nuclear DNA content (genome size, C-value) by flow cytometry. Nine species of the genus Eranthis from different parts of their ranges have been studied. It was found that they are characterized by a pronounced interspecies differences in basic karyometric parameters and localization of conservative molecular cytogenetic markers – 45S и 5S rDNA. Based on the size, chromosome morphology, number and size of rDNA bands, it is possible to clearly identify chromosome pairs in most diploid species. A positive correlation has been found between the number of 5S rDNA bands and genome size. For some species, intraspecific polymorphism has been identified for some karyotype parameters, the nature of which requires further detailed study. It has been suggested that chromosomal rearrangements, polyploidy, and the variation of number of repetitive sequences, including ribosomal genes, played a key role in the evolution of karyotype Eranthis. A comparative study of karyotypes for Halerpestes sarmentosa and H. salsuginosa has also been conducted. The differences of these species by karyomorphological parameters are shown. The results of the study were published in PhytoKeys (Q2, IF – 1.635). A metabolomic profile of water-ethanol extracts from the leaves of Eranthis longistipitata collected in natural populations of Kyrgyzstan was studied for the first time. Chemical compounds were analyzed by liquid chromatography with high-resolution mass spectrometry using advanced high-precision equipment. This method was chosen since it does not require a large amount of plant material, which is relevant for the study of endemic plants of the genus Eranthis. Data were obtained on the composition of the total metabolite pool of Eranthis longistipitata leaves (more than 160 compounds, of which 72 were identified as a class and 58 as an individual compound), the presence of furochromones and coumarins was detected, and data on the composition of the flavonoid component of the profile were corrected. The developed method for identifying substances in the leaves of Eranthis longistipitata will allow further phytochemical and chemotaxonomic studies of plant species of the genus Eranthis. The study of the metabolome in the genus Eranthis is another attempt to use advanced chromatographic methods to analyze and characterize both the metabolome and secondary metabolites on a poorly studied taxonomic group. The phytochemistry of the representatives of this genus is poorly studied. The analysis of literary sources showed that triterpene glycosides, saponins and their biological activity were studied for Eranthis cilicica species. A flavonoid complex was also studied in some representatives of the genus Eranthis. However, chromones found in plants of this genus, and coumarins, another group of benzo-α-pyrone derivatives, are of greatest interest. In contrast to other groups of compounds of the Eranthis species, which are often found in plants and are sufficiently studied, the limited distribution of chromones and coumarins and their pharmacological activity are of interest. For example, coumarins found in E. longistipitata exhibit antioxidant, antitumor, and antiapoptotic activities. Chromone derivatives show anti-inflammatory, antiviral, antitumor and antispasmodic effects and are used as antioxidants. In addition, due to their photochemical properties, they can be used as fluorescent labels in biochemical experiments and clinical medicine. The study of plant species containing chromones and coumarins, which are limited in the plant world, will provide new sources of useful pharmacologically active substances. The results obtained can also contribute to the study of the taxonomy of the fam. Ranunculaceae, since, in terms of phytochemical features, Eranthis is closer to the species from the genus Actae, which also synthesize chromones during their vital activity. The study results were published in 2 scientific papers in the International Journal of Molecular Sciences (Q1, IF – 5.92) and Plants-Basel (Q1, IF – 3.94).

 

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Annotation of the results obtained in 2019
During the taxonomical treatment Ranunculaceae species for the Himalaya flora the typification of all known Aquilegia taxa names from the region including species which have been described by John Royle and all the rest outstanding researchers of Pan-Himalaya flora was performed. For typification, we examined herbarium material and/or digital images deposited at BM, CAL, E, K, LIV, KHD, LE, and P (herbarium codes according to Thiers 2020 +). Furthermore, field works in Himalayas, to evaluate variation of morphological characters are provided. Lectotypes and epitypes are designated in accordance with the relevant articles of the International Code of Nomenclature for algae, fungi, and plants (Turland et al., 2018). The relevant information, indicated in the protologues (“Protologue citation”), is provided here for all the names. All specimens are cited in full; most of them were assigned the barcode numbers following the herbarium codes. As a result of the work one article have published in the Phytotaxa (Erst et al., 2020c), the second one prepared for submission to the same journal. Performed Aquilegia taxonomical revision for the Pan-Himalaya territory has indicated that new endemic species could still be found in some Asia mountainous regions. The diversity of Aquilegia species in India and Pakistan and Nepal is higher than previously thought, and the Himalayan region can be considered a continuation of the Central Asian center of diversity of the genus. As a result new species for science Aquilegia bashahrica and new hybrid Aquilegia × emodi from North-West Himalaya were described and illustrated. In addition, A. lactiflora was find in India and Pakistan, A. rockii was found in Nepal, and A. kareliniana was excluded from flora of India. In addition to the description of new taxa, the article provides an identification key for Aquilegia species from the north-west Himalayas and discusses diagnostic features, including those that were not previously used to distinguish taxa. The revision results are presented in the Phytotaxa (Erst et al., 2020a). The Siberian Eranthis revision in preliminary scientific research has revealed that E. sibirica still now is not typificated. In order to stabilize the use of the name in the sense in which it was used before and is now used, lectotyping was carried out. During the work herbarium material deposited at, G, LE, LECB, LINN and MW (herbarium codes according to Thiers 2020 +) were examined. The results are presented in the Phytotaxa (Erst et al., 2020b). Apart from conventional Sanger’s sequencing during the project realization an approach based on NGS (next generation sequencing) was used. RADseq (Restriction site Associated DNA Sequencing) was used for Halerpestes species research. Double-digest RAD-sequencing was utilized to generate phylogenomic data, both on fresh and old herbarium material of 23 samples. Delphinium was chosen as the outgroup to understanding phylogenetic relationships within Halerpestes and amongst some of its close relatives. The method produced high-quality phylogenomic data comprising millions of base pairs for each included sample. The sequencing library received was of high quality based on FastQC metrics. The data were processed through the ipyrad pipeline and phylogenetic inference was conducted both in raxml as well as in BEAST. Both ML and Bayesian approaches successfully reconstructed phylogenetic relationships within Halerpestes and across outgroups, and these relationships were generally highly supported. The genus Halerpestes dividet in to major well supported clades (I – species from the Tibetian platae; II–II-1 – species from the North Asia and II-2 – species from the North and South American), each defined largely by geography. Eranthis phylogeny and divergence times. Maximum likelihood (ML) and Bayesian inference (BI) analyses recovered a monophyletic Eranthis, sister to Actaea. Eranthis includes two major clades (I – white flowered species and II – yellow flowered species) and four well-supported subclades (I-1 – East Tibetian platae; I-2 – North Asia; II-1 – West Tibetian platae; II-2 – Mediterranean), each defined largely by geography. Within subclade I-2, the Mongolian plateau taxa united together and were nested within Northeast Asian taxa. The results of integrated molecular dating and ancestral range reconstruction analyses indicate that Eranthis originated in East Asia, and then dispersed from east (East Asia) towards west (West Tibetian platae and Mediterranean regions) between 56 and 41 Ma.). Cytogenetic parameters of European and Central Asian Eranthis species have been investigated. We have established somatic chromosome numbers, ploidy levels, and set of basic karyological parameters: total chromosome length for each pair, arm ratio, centromeric index, total haploid length of chromosome set, relative chromosome length, mean chromosome length in set, karyotype asymmetry indices: coefficient of variation of chromosome length (CVCL), coefficient of variation of centromeric index (CVCI), mean centromeric asymmetry (MCA). We have determined the nuclear DNA content via flow cytometry. We established the clear differences among chromosome sets. The differences between karyotypes are related to the types of heterobrachial chromosomes, their length dimensions and centromeric indexes, total haploid length of chromosome set and nuclear DNA content, coefficients of interchromosomal (CVCL) and intrachromosomal (MCA, CVCI) asymmetries. A comparative study of the parameters above, in different species and populations of the species, must assume across-species polymorphism and evolution of karyotypes. We have determined nuclear DNA content via flow cytometry of Aquilegia ecalcarata, A. flavescense, A. hebeica, A. laramiensis, A. semicalcarata, A. sibirica, A. yabeana. The values varied from 2С = 0.78 ± 0.02 pg in Aquilegia hebeica to 2С = 0.92 ± 0.03 pg in Aquilegia flavescense. As long as all investigated specimens had somatic chromosome number 2n = 14, we assumed that the evolution in the genus Aquilegia was associated with repetitive DNA variations.

 

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Annotation of the results obtained in 2020
During the taxonomical treatment Ranunculaceae species for the Himalaya flora the typification of all known Aquilegia taxa names from the region was performed. For typification, we examined herbarium material and/or digital images deposited at BM, CAL, E, K, LIV, KHD, LE, and P (herbarium codes according to Thiers 2020 +). Lectotypes and epitypes are designated in accordance with the relevant articles of the International Code of Nomenclature for algae, fungi, and plants (Turland et al., 2018). The relevant information, indicated in the protologues (“Protologue citation”), is provided here for all the names. All specimens are cited in full; most of them were assigned the barcode numbers following the herbarium codes. As a result of the work one article have published in the Phytotaxa (Erst et al., 2021). The goldthread genus Coptis includes 15 species disjunctly distributed in eastern Asia and North America. Here, we provide a dated phylogeny for the genus with all 15 species. Our results indicate that Coptis contains two strongly supported clades (I and II). Clade I consists of subg. Coptis and sect. Japonocoptis of subg. Metacoptis; clade II composes sect. Japonocoptis of subg. Metacoptis. Central leaflet base, sepal shape, and petal blade carry a strong phylogenetic signal in Coptis, while leaf type, sepal and petal color, and petal shape exhibit relatively higher levels of evolutionary flexibility. Our dating and biogeographic analyses indicate that a vicariance event between Japan-North America occurred in the middle Miocene, resulting in the split of Coptis and its sister group. Subsequently, a colonization event occurred at 9.55 Ma from Japan to mainland China. Both vicariance and dispersal events have played important roles in shaping the current distribution and endemism of Coptis, likely resulting from eustatic sea-level changes, mountain formation processes and an increasing drier and cooler climate from the middle Miocene onwards. The evolutionary history of organisms with poor dispersal abilities usually parallels geological events. Collisions of the Indian and Arabian plates with Eurasia greatly changed Asian topography and affected regional and global climates as well as biotic evolution. However, the geological evolution of Asia related to these two collisions remains debated. Here, we used Eranthis, an angiosperm genus with poor seed dispersal ability and a discontinuous distribution across Eurasia, to shed light on the orogenesis of the Qinghai–Tibetan, Iranian and Mongolian Plateaus. Our phylogenetic analyses show that Eranthis comprises four major geographical clades: east Qinghai–Tibetan Plateau clade (I-1), North Asian clade (I-2), west Qinghai–Tibetan Plateau clade (II-1) and Mediterranean clade (II-2). Our molecular dating and biogeographic analyses indicate that within Eranthis, four vicariance events correlate well with the two early uplifts of the Qinghai–Tibetan Plateau during the Late Eocene and the Oligocene–Miocene boundary and the two uplifts of the Iranian Plateau during the Middle and Late Miocene. The origin and divergence of the Mongolian Plateau taxa are related to the two uplifts of the Mongolian Plateau during the Middle and Late Miocene. Additionally, our results are in agreement with the hypothesis that the central part of Tibet only reached an altitude of less than 2.3 km at approximately 40 Ma. This study highlights that organismal evolution could be related to the formation of the three great Asian plateaus, hence contributing to the knowledge on the timing of the key tectonic events in Asia. A cytogenetic approach keeps being relevant to the systematics and phylogeny problem solving, although the molecular genetic methods are widely used. The comparative karyotype analysis as a part of the integrative taxonomic approach is used successfully along with morphological, molecular genetic, phytochemical, and other methods to study plants of different taxonomic groups, including fam. Ranunculaceae Juss. Karyotype formulas of Halerpestes distributed in Russia were first studied. They were 2n = 2x = 16 = 8m + 4sm + 4st in H. sarmentosa, and 2n = 2x = 48 = 24m + 6sm + 18st in H. salsuginosa. The chromosome set of Eranthis cilicica, Turkish endemic species, was studied and its specificity as compared to other related species was established. 5S rRNA genes were first localized on chromosomes of Eranthis byunsanensis, E. pinnatifida, E. stellata, E. tanhoensis, and E. cilicica. The oligonucleotide 5.8S rDNA probe for Ranunculaceae was originally designed and tested. The composition of flavonoids in 70% water-ethanol extracts from the leaves of E. longistipitata was studied by HPLC LC-MS, NMRs for the first time. Mass spectrometry revealed 16 flavonoid compounds. Substances belong to the following groups of flavonoids: flavonols, flavans, chalcones, flavanones and flavons. The greatest variety is observed among flavonols. Eight substances of this group have been identified: two free aglycones (quercetin and kaempferol), four quercetin glycosides (hyperoside, rutin, hirsutrin and quercetin 3-sambucoside) and two kaempferol glycosides (juglanin and tripolin). In the leaves of E. longistipitata, two flavans ((+) - catechin and auriculoside), two hydroxyflavanones (6-methoxytaxifolin and aromadendrin), and one flavone - luteolin C-glycoside (carlinoside) were indicated. Dihydroxychalcones aspalatin, phloridzin, and phloretin were also found in leaf extracts. As a result of the analysis, it was concluded that the diversity of the flavonoid compound of E. longistipitata leaves is probably due to external ecological and geographical factors and is associated with the growing season and adaptive functions.

 

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