Chloroplast DNA Sequences Research Articles

Molecular evidence for the status of Bidens connata Muhl. ex Willd. and B. decipiens Warnst. in the old and new World

Earlier, we have established that the European blackjack, which in many literary sources is cited as an invasive North American Bidens connata, was described by Carl Warnstorf back in 1895 as B. decipiens and had a hybrid origin (B. frondosa × B. cernua). In this study, we continue to compare the genomes of B. connata and B. decipiens by molecular genetics and cytological methods. The objects are the F1 offsprings of B. frondosa, B. connata, and B. cernua collected in 2018 from Minnesota and Wisconsin (USA), grown from seeds in the greenhouse conditions of N.V. Tsitsin Main Botanical Garden of Russian Academy of Sciences, as well as samples of B. decipiens, B. frondosa and B. cernua collected from Eastern Europe (Belarus and European Russia). The nucleotide sequences of nuclear (ITS 1–2) and chloroplast (trnL– trnF and rpl32–trnL) DNA were studied. Analysis of the ITS 1–2 site showed that B. connata individuals of North America are not hybrids. Analysis of the chloroplast DNA regions confirmed that both taxa, B. connata and B. decipiens, are evolutionarily close to B. cernua.
 Bangladesh J. Plant Taxon. 28(1): 1-10, 2021 (June)

Phylogeny and historical biogeography of the pantropical genus Parkia (Leguminosae, Caesalpinioideae, mimosoid clade)

Parkia R.Br. (Leguminosae, Caesalpinioideae, mimosoid clade) is a pantropical genus with approximately 35 recognized species in three taxonomic sections (Parkia, Platyparkia and Sphaeroparkia), distributed widely in tropical forests and savannas in South and Central America, Africa-Madagascar and the Indo-Pacific region. In this study, phylogenetic analyses (Maximum Likelihood and Bayesian Inference), ancestral area and habitat estimations were performed using chloroplast (matK, trnL, psbA-trnH and rps16-trnQ) and nuclear (ITS/18S/26S) DNA sequences for the purpose of testing the monophyly of Parkia and inferring the geographic origin of the genus and times of divergence of the various lineages. This enabled investigation of factors that may have influenced its diversification in both hemispheres. Our results support the monophyly of the genus. A fossil-calibrated Bayesian analysis dated the Parkia crown node to the Miocene (at c. 18.85 Ma). Biogeographic analysis reconstructed an origin in the lowlands rainforests (terra firme) in Amazonia with subsequent radiation in the Neotropical region from the Miocene onwards, with dispersion events as far as Central America, and the Atlantic Forest and the cerrado of Brazil. A single dispersion from the Neotropics to the Paleotropics is hypothesised, with subsequent smaller radiations in Africa-Madagascar and the Indo-Pacific (crown ages 3.79 and 5.15 Ma respectively). Factors that may have influenced the radiation and speciation of Parkia include the elevation of the Andes (especially in the Miocene), and more recently the closing of the Panama gap in Neotropics, the climatic fluctuations of the Pleistocene influenced the diversification of species on both continents. The elevation of the Sunda Shelf in Indo-Pacific region during the last glacial maximum (LGM) appears to be the main driving force for speciation in that region. In Africa, the low number of species may be related to extinction processes.

Genomic diversity of 39 samples of Pyropia species grown in Japan.

Some Pyropia species, such as nori (P. yezoensis), are important marine crops. We conducted a phylogenetic analysis of 39 samples of Pyropia species grown in Japan using organellar genome sequences. A comparison of the chloroplast DNA sequences with those from China showed a clear genetic separation between Japanese and Chinese P. yezoensis. Conversely, comparing the mitochondrial DNA sequences did not separate Japanese and Chinese P. yezoensis. Analysis of organellar genomes showed that the genetic diversity of Japanese P. yezoensis used in this study is lower than that of Chinese wild P. yezoensis. To analyze the genetic relationships between samples of Japanese Pyropia, we used whole-genome resequencing to analyze their nuclear genomes. In the offspring resulting from cross-breeding between P. yezoensis and P. tenera, nearly 90% of the genotypes analyzed by mapping were explained by the presence of different chromosomes originating from two different parental species. Although the genetic diversity of Japanese P. yezoensis is low, analysis of nuclear genomes genetically separated each sample. Samples isolated from the sea were often genetically similar to those being farmed. Study of genetic heterogeneity of samples within a single aquaculture strain of P. yezoensis showed that samples were divided into two groups and the samples with frequent abnormal budding formed a single, genetically similar group. The results of this study will be useful for breeding and the conservation of Pyropia species.

Genetic diversity patterns in Phragmites australis populations in straightened and in natural river sites in Lithuania

Like in many other parts of the world, Phragmites australis distribution and abundance are changing in Lithuania because of the impact of human activities on aquatic habitats. We studied P. australis genetic diversity patterns and the effect of hydrographic modifications, introduced in the mid-late twentieth century with great impact on Lithuanian landscape. The genetic diversity was studied using chloroplast DNA sequences and nuclear microsatellite markers in natural and straightened river stretches for water regulation. We found haplotypes M and L and their variants in the studied populations. The analysis of microsatellites revealed high genetic diversity within populations and significant structure both at the population and river level. We did not find differences in the distribution of genetic diversity between populations growing in natural and in straightened river stretches; however, at the local level, 5 populations in straightened river sites had higher genetic diversity values than populations in nearby natural sites within the same river, confirming seed establishment in disturbed habitats. Our results demonstrate that anthropogenic river modifications have had an impact on the genetic diversity of P. australis populations; however, disturbance is not the only factor that affects genetic diversity recruitment and dynamics in new P. australis stands.

Phylogeny and Sequence Analysis of Sulla species Based on Intergenic Spacers trnL-trnF

Chloroplast DNA (cpDNA) sequence variations are broadly used to investi-gate interspecific relationships among plants. The trnL-trnF spacer is located in the large single-copy region of the chloroplast genome. The objective of this study was to evaluate the level of genetic variability between the Algeri-an and Tunisian species Hedysarum pallidum (North African endemic spe-cies) and H. capitatum. The phylogenetic relationships among the studied species were investigated by means of maximum Likehood analysis of se-quence polymorphism from chloroplast trnL-trnF spacer DNA; the tree parti-tioned the studied species in two clades. In addition, some specific variations loci- were identified between the species. The observed variations suggest considerable differentiation among cpDNA which could be useful to identify species.

Diversification in both the floral morphology and chemistry in two daciniphilous orchid ecotypes in Borneo

Morphological and floral chemical components are both important cues used by plants to attract pollinators. Two local ecotypes named ‘Nabawan’ and ‘Apin-Apin’ produced by the tropical orchid, Bulbophyllum ecornutum subspecies verrucatum, exhibit different morphology and scents to attract males of certain Bactrocera and Zeugodacus fruit fly species in the tribe Dacini as potential pollinators. Flowers of the Nabawan-ecotype possess a narrow-shaped lip (= labellum) structure and emit a characteristic fragrance of raspberry ketone along with smaller amounts of rhododendrol, whereas those of Apin-Apin-ecotype possess a broad-shaped lip and emit zingerone and zingerol as major components. Nevertheless, the structure of stiff hamulus appears similar and acts as a ‘crowbar’ during pollinarium removal by a fruit fly, indicating the basic mechanics of the movable see-saw lip structure to trap a fly is essentially the same. The changes in both morphology and chemistry, despite no significant difference in chloroplast DNA sequences, shown by the two ecotypes, might have occurred as part of a coevolutionary process involving both the Dacini fruit fly pollinators and the Bulbophyllum orchids in their natural habitats.

Identification of Cannabis Sativa L. Based on rbcL Sequence.

Objective To assess the feasibility of the rbcL sequence of chloroplast DNA as a genetic marker to identify Cannabis sativa L. Methods The rbcL sequences in 62 Cannabis sativa L. samples, 10 Humulus lupulus samples and 10 Humulus scandens DNA samples were detected, and 96 rbcL sequences of the Cannabaceae family were downloaded from Genbank. Sequence alignment was performed by MEGA X software, the intraspecific and interspecific Kimura-2-Parameter （K2P） genetic distances were calculated, and the system clustering tree was constructed. Results The rbcL sequence length acquired by sequencing of Cannabis sativa L. and Humulus scandens were 617 bp and 649 bp, respectively, and two haplotypes of Cannabis sativa L. were observed in the samples. The BLAST similarity search results showed that the highest similarity between the sequences acquired by sequencing and Cannabis sativa L. rbcL sequences available from Genbank was 100%. The genetic distance analysis showed that the maximum intraspecific genetic distance （0.004 9） of Cannabis sativa L. was less than the minimum interspecific genetic distance （0.012 9）. The results of median-joining network and system clustering tree analysis showed that Cannabis sativa L. and other members of the Cannabaceae family were located in different branches. Conclusion The rbcL sequence could be used as a DNA barcode for identifying Cannabis sativa L., and combined with comparative analysis of the rbcL sequence and system cluster analysis could be a reliable and effective detection method for Cannabis sativa L. identification in forensic investigation.

Global phylogeography of a pantropical mangrove genus Rhizophora

Rhizophora is a key genus for revealing the formation process of the pantropical distribution of mangroves. In this study, in order to fully understand the historical scenario of Rhizophora that achieved pantropical distribution, we conducted phylogeographic analyses based on nucleotide sequences of chloroplast and nuclear DNA as well as microsatellites for samples collected worldwide. Phylogenetic trees suggested the monophyly of each AEP and IWP lineages respectively except for R. samoensis and R. × selala. The divergence time between the two lineages was 10.6 million years ago on a dated phylogeny, and biogeographic stochastic mapping analyses supported these lineages separated following a vicariant event. These data suggested that the closure of the Tethys Seaway and the reduction in mangrove distribution followed by Mid-Miocene cooling were key factors that caused the linage diversification. Phylogeographic analyses also suggested the formation of the distinctive genetic structure at the AEP region across the American continents around Pliocene. Furthermore, long-distance trans-pacific dispersal occurred from the Pacific coast of American continents to the South Pacific and formed F1 hybrid, resulting in gene exchange between the IWP and AEP lineages after 11 million years of isolation. Considering the phylogeny and phylogeography with divergence time, a comprehensive picture of the historical scenario behind the pantropical distribution of Rhizophora is updated.

Refugia during the last glacial period and the origin of the disjunct distribution of an insular plant

AbstractAimAlthough many phylogeographic studies have focused on continental refugia, the roles of islands as refugia have long been overlooked. In this study, we conducted phylogeographic studies of a plant with insular distribution, Microtropis japonica, to elucidate the potential roles of islands as refugia during recent glacial periods. Specifically, we test the hypothesis that two remote groups of Japanese islands (the Izu and Ryukyu Islands) were refugia for M japonica.LocationThe Izu and Ryukyu Islands of Japan and their adjacent areas including the Japanese mainland and Taiwan.TaxonMicrotropis japonica (Celastraceae).MethodsA total of 363 individuals were sampled from 44 populations of M japonica. To elucidate population genetic differentiation and demographic change in M japonica, we performed molecular variance and mismatch distribution analyses based on chloroplast DNA (cpDNA) sequence variations and Bayesian clustering and gene flow estimates based on nuclear single‐nucleotide polymorphism (SNP) data. In addition, to examine distribution changes, ecological niche modelling using the maximum entropy method was used to determine suitable habitats existing currently or during the last glacial maximum, using occurrence data and six bioclimatic variables.ResultsBoth cpDNA and nuclear SNP data showed genetic differentiation between two disjunct regions. However, cpDNA data indicated genetic differentiation within the Ryukyu Islands but not within the Izu Islands, while nuclear SNP data indicated genetic differentiation within both island groups. Ecological niche modelling showed that both the Izu and Ryukyu archipelagos had contained some islands with a climate compatible with modern M japonica climate envelopes despite climate oscillations.Main conclusionsIslands within both the Izu and Ryukyu archipelagos may have supported M japonica populations throughout historical climate oscillations. The current disjunct distribution of M japonica strongly reflects its refugial locations during the last glacial period and the subsequent range expansion. Islands were important refuges, contributing to the modern disjunct distribution of M japonica.

Reinstatement of the New Caledonian endemic fern Blechnum deplanchei as distinct from B. opacum (Blechnaceae)

ABSTRACT Blechnum opacum is a small forest fern indigenous to New Caledonia and Vanuatu. After examining the considerable morphological variation in New Caledonian populations, along with phylogenetic analysis of chloroplast DNA sequences, we conclude that B. deplanchei should be reinstated for some of the plants in New Caledonia. Blechnum deplanchei is readily recognisable, with pinnatisect fronds that are usually larger, pinnae that are numerous and oblong, and with the distal lamina segments reduced in size. Based on two chloroplast loci, the three sampled populations of B. deplanchei comprised a distinct and well-supported lineage. The genetic and morphological distinctiveness of B. deplanchei was maintained over a reasonably large area, including areas of broad sympatry with B. opacum. Blechnum deplanchei is largely confined to the mountains in southern Grande Terre, in areas underlain by ultramafic substrates. It generally occurs at higher elevations, and is assessed with a conservation status of Near Threatened. Even with the recognition of B. deplanchei, B. opacum remains a variable species. A few plants are morphologically intermediate. We provisionally retain them in B. opacum, but they require further investigation. The morphologically intermediate samples in the phylogenetic analyses were the most distantly related among the ingroup, contributing to the paraphyly of B. opacum with respect to B. deplanchei. The morphologically intermediate plants are unlikely to be first generation hybrids or allopolyploids, given their genetic distinctiveness, the regularity and comparable size of their spores, and because some occur far to the north of the distribution of B. deplanchei; furthermore, Vanuatu plants have a similar morphology. It is possible that they may simply be unusually large plants of B. opacum, or a distinct, third species. Resolving their status requires genetic analysis of additional samples from northern Grande Terre and Vanuatu. With the circumscription adopted here, we assess B. opacum as Least Concern.

Lastreopsis abscondita (Dryopteridaceae), a new fern species endemic to New Caledonia

ABSTRACT The fern genus Lastreopsis has been recently re-circumscribed to include about 19 species. It occurs from southern Asia through the Pacific to the Neotropics, with Australasia having the most species. We here describe a new species endemic to New Caledonia’s Grande Terre, L. abscondita. Morphological comparisons and phylogenetic analyses of chloroplast DNA sequences show that L. abscondita resembles and is closely related to L. vieillardii, also endemic to New Caledonia. From L. vieillardii, L. abscondita differs most noticeably in being much scalier, particularly on its rachis and costae, and in having larger scales, and larger, more-divided fronds. Chloroplast DNA sequences distinguish samples of L. abscondita as a clade, but indicate there is substantial genetic variation within the narrowed circumscription of L. vieillardii such that it is paraphyletic; this should be further investigated. Lastreopsis abscondita is known from only two sites, both at high elevation on non-ultramafic substrates on Grande Terre. We propose an IUCN conservation status of Vulnerable for L. abscondita, particularly because of the small number of sites and limited area of occupancy. The specific epithet alludes to the fact that this large fern, with fronds over 1.5 m long, has been overlooked until now. This is another indication that the ferns of New Caledonia warrant further study, with more discoveries likely to be made.

Targeted enrichment of novel chloroplast-based probes reveals a large-scale phylogeny of 412 bamboos

BackgroundThe subfamily Bambusoideae belongs to the grass family Poaceae and has significant roles in culture, economy, and ecology. However, the phylogenetic relationships based on large-scale chloroplast genomes (CpGenomes) were elusive. Moreover, most of the chloroplast DNA sequencing methods cannot meet the requirements of large-scale CpGenome sequencing, which greatly limits and impedes the in-depth research of plant genetics and evolution.ResultsTo develop a set of bamboo probes, we used 99 high-quality CpGenomes with 6 bamboo CpGenomes as representative species for the probe design, and assembled 15 M unique sequences as the final pan-chloroplast genome. A total of 180,519 probes for chloroplast DNA fragments were designed and synthesized by a novel hybridization-based targeted enrichment approach. Another 468 CpGenomes were selected as test data to verify the quality of the newly synthesized probes and the efficiency of the probes for chloroplast capture. We then successfully applied the probes to synthesize, enrich, and assemble 358 non-redundant CpGenomes of woody bamboo in China. Evaluation analysis showed the probes may be applicable to chloroplasts in Magnoliales, Pinales, Poales et al. Moreover, we reconstructed a phylogenetic tree of 412 bamboos (358 in-house and 54 published), supporting a non-monophyletic lineage of the genus Phyllostachys. Additionally, we shared our data by uploading a dataset of bamboo CpGenome into CNGB (https://db.cngb.org/search/project/CNP0000502/) to enrich resources and promote the development of bamboo phylogenetics.ConclusionsThe development of the CpGenome enrichment pipeline and its performance on bamboos recommended an inexpensive, high-throughput, time-saving and efficient CpGenome sequencing strategy, which can be applied to facilitate the phylogenetics analysis of most green plants.

Parentage analysis of tea cultivars in Japan based on simple sequence repeat markers.

Tea cultivars have been bred by individual selection of landraces and by crossbreeding, but the validation of the parentage is limited. In this study, we performed parentage analysis of 79 tea cultivars in Japan based on SSR markers to confirm or identify the parent-offspring relationships among them. The effectiveness of nine SSR markers for parentage analysis was validated by comparing them to the existing cleaved amplified polymorphic sequence markers. The former markers were detectable more alleles than the latter. Simulation of parentage analysis of the tea cultivars predicted biparental origins for 12 cultivars (‘Houshun’, ‘Mie ryokuhou no. 1’, ‘Surugawase’, ‘Tenmyo’, ‘Yamanoibuki’, ‘Harumidori’, ‘Koushun’, ‘Minekaori’, ‘Okumusashi’, ‘Saemidori’, ‘Sofu’, and ‘Toyoka’), in the first five of which candidate parents of yet-to-be-defined pedigree were newly identified. Comparisons of a total of 41 SSR genotypes confirmed the newly-identified parentages of ‘Asahi’ for ‘Tenmyo’, ‘Rokurou’ for ‘Houshun’, ‘Surugawase’, and ‘Yamanoibuki’, and ‘Yamatomidori’ for ‘Mie ryokuhou no. 1’. The maternity of seven cultivars out of the 12 was also confirmed with chloroplast DNA sequences. Uniparental origins were confirmed for 25 cultivars. This parentage analysis has improved our knowledge of tea pedigrees and will aid in the development of new cultivars.

Identification of three independent fern gametophytes and Hymenophyllum wrightii f. serratum from Korea based on molecular data

Colonies of three independent gametophytes (one that is filamentous and two that are ribbon-like) without sporophytes occur in Gyeonggi-do, Gangwon-do, Gyeongsang-do, and Jeju-do, Korea. They have a moss-like appearance at first sight, with tiny plantlets and gemmae, and grow in cool, shaded, relatively deep dint places of large rocks, such as the small caves in high mountains, close to valleys. The gametophytes were identified based on morphological and molecular data by chloroplast DNA (cpDNA) sequence data (rbcL, rps4 gene and rps4-trnS intergenic spacer). Here, rbcL, rps4 gene and rps4-trnS intergenic spacer data of one independent gametophyte distributed in Korea have the same morphology, DNA sequence and monophyletic group as Crepidomanes intricatum from the eastern United States. They also share the same cpDNA data with Crepidomanes schmidtianum recently reported from Korea. The other independent gametophyte should be Hymenophyllum wrightii based on cpDNA data. The last one was presumed to be Pleurosoriopsis makinoi based on molecular data. The taxonomic status was confirmed to be the forma of Hymenophyllum wrightii through a revision of Hymenophyllum wrightii f. serratum based on molecular data.

SPInDel Analysis of the Non-Coding Regions of cpDNA as a More Useful Tool for the Identification of Rye (Poaceae: Secale) Species.

Secale is a small but very diverse genus from the tribe Triticeae (family Poaceae), which includes annual, perennial, self-pollinating and open-pollinating, cultivated, weedy and wild species of various phenotypes. Despite its high economic importance, classification of this genus, comprising 3–8 species, is inconsistent. This has resulted in significantly reduced progress in the breeding of rye which could be enriched with functional traits derived from wild rye species. Our previous research has suggested the utility of non-coding sequences of chloroplast and mitochondrial DNA in studies on closely related species of the genus Secale. Here we applied the SPInDel (Species Identification by Insertions/Deletions) approach, which targets hypervariable genomic regions containing multiple insertions/deletions (indels) and exhibiting extensive length variability. We analysed a total of 140 and 210 non-coding sequences from cpDNA and mtDNA, respectively. The resulting data highlight regions which may represent useful molecular markers with respect to closely related species of the genus Secale, however, we found the chloroplast genome to be more informative. These molecular markers include non-coding regions of chloroplast DNA: atpB-rbcL and trnT-trnL and non-coding regions of mitochondrial DNA: nad1B-nad1C and rrn5/rrn18. Our results demonstrate the utility of the SPInDel concept for the characterisation of Secale species.

A New Species of Monrosia (Polygalaceae) from Argentina, and Revision of this Genus Endemic to the Southern Andes

Abstract—The recently resurrected monotypic genus Monrosia (Polygalaceae) is endemic to the Argentinian provinces of Catamarca to San Juan in the southern Andes. We here describe and illustrate Monrosia sanjuanensis, a new species endemic to the province of San Juan and the second species of the genus, and revise the genus. Monrosia and its two species are described and analysed in a phylogenetic context, using nuclear and chloroplast DNA sequences. A distribution map, illustration, photos of the morphological structures of the two species, and taxonomic key are provided. We designate a lectotype for the name M. pterolopha.

Genome-wide RAD sequencing resolves the evolutionary history of serrate leaf Juniperus and reveals discordance with chloroplast phylogeny

Juniper (Juniperus) is an ecologically important conifer genus of the Northern Hemisphere, the members of which are often foundational tree species of arid regions. The serrate leaf margin clade is native to topologically variable regions in North America, where hybridization has likely played a prominent role in their diversification. Here we use a reduced-representation sequencing approach (ddRADseq) to generate a phylogenomic data set for 68 accessions representing all 22 species in the serrate leaf margin clade, as well as a number of close and distant relatives, to improve understanding of diversification in this group. Phylogenetic analyses using three methods (SVDquartets, maximum likelihood, and Bayesian) yielded highly congruent and well-resolved topologies. These phylogenies provided improved resolution relative to past analyses based on Sanger sequencing of nuclear and chloroplast DNA, and were largely consistent with taxonomic expectations based on geography and morphology. Calibration of a Bayesian phylogeny with fossil evidence produced divergence time estimates for the clade consistent with a late Oligocene origin in North America, followed by a period of elevated diversification between 12 and 5 Mya. Comparison of the ddRADseq phylogenies with a phylogeny based on Sanger-sequenced chloroplast DNA revealed five instances of pronounced discordance, illustrating the potential for chloroplast introgression, chloroplast transfer, or incomplete lineage sorting to influence organellar phylogeny. Our results improve understanding of the pattern and tempo of diversification in Juniperus, and highlight the utility of reduced-representation sequencing for resolving phylogenetic relationships in non-model organisms with reticulation and recent divergence.

<p><strong><em>Ophioglossum chaloneri</em></strong><strong>: A new species of <em>Ophioglossum </em>(Ophioglossaceae: Pteridophyta) from India</strong></p>

In the present study, we describe Ophioglossum chaloneri as a new species from the Hurdulu forest area, near Ranchi (Jharkhand, India), on the basis of morphological, palynological, cytological, and molecular analyses. The new species can be distinguished from all other congeners of genus Ophioglossum in having their unusually small linear rhizome with a long stalk of trophophore, a sporophore arising from a distance from the base of the trophophore hence not always adnate or appearing inserted at the base. Triradiate, round spores show small tubercles towards the proximal pyramidal area and irregular smooth elevated exine striations on both the proximal and distal faces. The ventral face of the trophophore is unusually thin all around margin thereby showing thin whitish margin from the lower side, a situation that may be due to lack of chloroplast all around the margin at the lower face of the trophophore. This feature is not observed in any other species of the genus. Molecular study based on three chloroplast DNA sequences (cpDNA) rbcL, trnL-F, and psbA-trnH also supports O. chaloneri as a distinct species.

Evaluation of Genetic Diversity by DNA Barcoding of Local Lotus Populations from Thua Thien Hue Province

Background: DNA barcoding is a relatively new method of identifying plant species using short sequences of chloroplast DNA. Although there is a large number of studies using barcoding on various plant species, there are no such studies in the genus Nelumbo. Methods: Three chloroplast DNA regions (rbcL, matK, trnH-psbA) were tested for their suitability as DNA barcoding regions of thirty three lotus samples which were collected in Thua Thien Hue province, Vietnam. Universal primers were used and sequenced products were analyzed using Minimum Evolution method in the MEGA 7.0 program.Result: We did not observe high variability in nucleotide sequences within the rbcL region (0.135%). White Nelumbo, while, the most encoding matK (8.013%) and variable trnH-psbA (with different number of repeating regions TAAAA) intergenic regions was the most useful for Nelumbo barcoding. Individual application of the studied regions did not provide the expected results. None of the regions used in the study allowed the division of white and pink lotus varieties of N. nucifera specie according to the adopted classification of the genus Nelumbo. The results confirm that the use of matK, rbcL and trnH-psbA or combine all three regions together is insufficient for DNA barcoding in white and pink lotus varieties of N. nucifera specie and better discrimination within the genus Nelumbo. Our results also indicate the necessity of using a different region. All of the new sequences have been deposited in GeneBank under the following accession numbers: rbcL (MN011708 to MN068956); matK (MN011719 to MN068978) and trnH-psbA (MN011730 to MN086252).

Phylogenetic relationships and status of taxa of Pulsatilla uralensis and P. patens s.str. (Ranunculaceae) in north-eastern European Russia.

We studied the allopatric complex Pulsatilla patens (L.) Mill. s.lat. (Ranunculaceae) in north-eastern European Russia and the Urals. In this region, there are two kinds of P. patens with different perianth colours in monochrome and polychrome populations. To clarify their taxonomic boundaries, we used the sequences of chloroplast DNA (rbcL and matK) and nuclear DNA (ITS2), in addition to morphological characteristics. The combination of three markers (rbcL+matK+ITS2) was found to be the most effective for phylogenetic resolution. The samples of two morphologically-different taxa P. uralensis and P. patents s.str. were shown to form a single clade on the phylogenetic tree. Based on the molecular phylogenetic analysis, we were not able to unequivocally prove the independent existence of P. uralensis.