DNA barcoding of selected UAE medicinal plant species: a comparative assessment of herbarium and fresh samples

It is commonly difficult to extract and amplify DNA from herbarium samples as they are old and preserved using different compounds. In addition, such samples are subjected to the accumulation of intrinsically produced plant substances over long periods (up to hundreds of years). DNA extraction from desert flora may pause added difficulties as many contain high levels of secondary metabolites. Herbarium samples from the Biology Department (UAE University) plant collection and fresh plant samples, collected from around Al-Ain (UAE), were used in this study. The three barcode loci for the coding genes matK, rbcL and rpoC1-were amplified. Our results showed that T. terresteris, H. robustum,T. pentandrus and Z. qatarense were amplified using all three primers for both fresh and herbaium samples. Both fresh and herbarium samples of C. comosum, however, were not amplified at all, using the three primers. Herbarium samples from A. javanica, C. imbricatum, T. aucherana and Z. simplex were not amplified with any of the three primers. For fresh samples 90, 90 and 80% of the samples were amplified using matK, rbcL and rpoC1, respectively. In short, fresh samples were significantly better amplified than those from herbarium sources, using the three primers. Both fresh and herbarium samples from one species (C. comosum), however, were not successfully amplified. It is also concluded that the rbcL regions showed real potentials to distinguish the UAE species under investigation into the appropriate family and genus.     

A report on identification of sequence polymorphism in barcode region of six commercially important <Emphasis Type="Italic">Cymbopogon</Emphasis> species

Cymbopogon

is an important member of grass family Poaceae, cultivated for essential oils which have greater medicinal and industrial value. Taxonomic identification of Cymbopogon species is determined mainly by morphological markers, odour of essential oils and concentration of bioactive compounds present in the oil matrices which are highly influenced by environment. Authenticated molecular marker based taxonomical identification is also lacking in the genus; hence effort was made to evaluate potential DNA barcode loci in six commercially important Cymbopogon species for their individual discrimination and authentication at the species level. Four widely used DNA barcoding regions viz., ITS 1 & ITS 2 spacers, matK, psbA-trnH and rbcL were taken for the study. Gene sequences of the same or related genera of the concerned loci were mined from NCBI domain and primers were designed and validated for barcode loci amplification. Out of the four loci studied, sequences from matK and ITS spacer loci revealed 0.46% and 5.64% nucleotide sequence diversity, respectively whereas the other two loci i.e., psbA-trnH and rbcL showed 100% sequence homology. The newly developed primers can be used for barcode loci amplification in the genus Cymbopogon. The identified Single Nucleotide Polymorphisms from the studied sequences may be used as barcodes for the six Cymbopogon species. The information generated can also be utilized for barcode development of the genus by including more number of Cymbopgon species in future.

     

Study on chloroplast DNA diversity of cultivated and wild pears (<Emphasis Type="Italic">Pyrus</Emphasis> L.) in Northern China

Eight pairs of chloroplast DNA (cpDNA) universal primers selected from 34 pairs were used to assess the genetic diversity of 132 pear accessions in Northern China. Among them, six amplified cpDNA fragments showed genetic diversity. A total of 24 variable sites, including 1 singleton variable site and 23 parsimony informative sites, as well as 21 insertion-deletion fragments, were obtained from the combined cpDNA sequences (5309–5535 bp). Two trnL-trnF-487 haplotypes, five trnL-trnF-413 haplotypes, five rbcL haplotypes, six trnS-psbC haplotypes, eight accD-psaI haplotypes and 12 rps16-trnQ haplotypes were identified among the individuals. Twenty-one haplotypes were identified based on the combined fragments. The values of nucleotide diversity (P_i), average number of nucleotide differences (k) and haplotype diversity (H_d) were 0.00070, 3.56408 and 0.7960, respectively. No statistical significance was detected in Tajima’s D test. Remarkably, the important cpDNA haplotypes and their representing accessions were identified clearly in this study. H_19 was considered as one of the ancient haplotypes and was a divergent centre. H_16 was the most common haplotype of the wild accessions. H_2 was the haplotype representing the most pear germplasm resources (46 cultivars and two wild Ussurian Pear accessions), followed by haplotype H_5 (30 cultivars, two wild Ussurian Pear accessions and four sand pears in outgroups) representing the cultivars ‘Dangshan Suli’ and ‘Yali’, which harbour the largest and the second largest cultivation areas in China. More importantly, this study demonstrated, for the first time, the supposed evolution routes of Pyrus based on cpDNA divergence in the background of pear phylogeny in Northern China.     

The earliest samples of <Emphasis Type="Italic">Hymenoscyphus albidus</Emphasis> vs. <Emphasis Type="Italic">H. fraxineus</Emphasis> in Estonian mycological herbaria

In 2006, the alien and pathogenic Hymenoscyphus fraxineus was differentiated by molecular methods from the long recognized saprotrophic H. albidus . Today, H. fraxineus seems to have replaced H. albidus in several countries, but the exact year of arrival of H. fraxineus in northern Europe is still debated. Investigations of herbaria specimens might help to ascertain it. Before the epidemic, H. albidus was not of significant interest to mycologists in the Baltic area and was not eagerly sampled and deposited. Nevertheless, the TAAM herbarium in Estonia holds 13 putative H. albidus specimens that were collected 1966–2006. Using newly developed species-specific PCR primers to differentiate between H. albidus and H. fraxineus, all available herbarium samples from TAAM were identified. In addition, the primers were also tested on pure cultures and ascocarps of H. fraxineus and H. albidus, symptomatic petioles of F. excelsior, pure cultures of 10 non-target fungal species, and 10 different soil samples. With the exception of the oldest specimen in TAAM from 1966 (collected in Lithuania), all herbarium samples were identified as H. fraxineus. The first record of H. fraxineus in Estonia dates back to 1997 and apparently represents the oldest record of the species in Europe. In spite of this, symptoms based on reliable observations were not detected until 2003 in Estonia. This temporal shift is difficult to explain. Possible reasons may represent (i) overlooking of symptoms during the first years; (ii) typical lag phase at the start of an epidemic; (iii) an abrupt shift towards higher virulence of H. fraxineus; or (iv) other environmental (climatic) factors. Closer investigations are needed to disentangle these possible reasons.     

Molecular phylogenetic analysis of key <Emphasis Type="Italic">Jatropha</Emphasis> species inferred from nrDNA ITS and chloroplast (<Emphasis Type="Italic">trn</Emphasis>L-F and <Emphasis Type="Italic">rbc</Emphasis>L) sequences

The genus Jatropha (Euphorbiaceae) contains species that are of significant economic and ornamental value. However, Jatropha breeding material is rather limited due to incomplete information regarding phylogenetic relationships among germplasm resources. Phylogenetic analyses were performed based on the internal transcribed spacer of nuclear ribosomal DNA (nrDNA ITS), two chloroplast regions (trnL-F and rbcL), and the combined (ITS+trnL-F+rbcL) dataset among twenty-five specimens representing six key Jatropha species. Phylogenetic relationships of Jatropha were well resolved between subgenus Curcas and subgenus Jatropha, and demonstrated the intermediate position of section Polymorphae among sections of both subgenera. Jatropha curcas and J. integerrima demonstrated a close phylogenetic relationship. The molecular data agreed with the morphological classification that recognized J. multifida and J. podagrica in sec. Peltatae. The distinct intraspecific divergence that occurred in J. curcas could be attributed to restricted gene flow caused by geographical isolation and different ecological conditions. Phylograms produced with trnL-F and rbcL sequence data suggested slow rates of sequence divergence among Jatropha spp., while the ITS gene tree had good resolution suggesting high genetic variation of ITS among Jatropha species.     

Molecular phylogenetic and biogeographical analysis of <Emphasis Type="Italic">Nitraria</Emphasis> based on nuclear and chloroplast DNA sequences

Based upon DNA sequences from six plastid regions (rbcL, psbB-psbH, trnL-trnF, rpS16, psbA-trnH, rpS16-trnK) and the internal transcribed spacer (ITS) region of nuclear ribosomal DNA, the phylogenetic relationships in the genus Nitraria and family Nitrariaceae are investigated by using methods of maximum parsimony, maximum likelihood, and Bayesian inference. Our study strongly supports the monophyly of Nitraria. Nitraria can be divided into four parts, namely, the N. sphaerocarpa group, N. retusa group, the N. roborowskii and N. tangutorum group, and a group consisting of N. schoberi, N. komarovii, N. sibirica, and N. billardieri. Ancestral area reconstruction using S-Diva shows that eastern Central Asia is most likely the place of origin, and then dispersals occurred to western Central Asia, Africa, and Australia.     

Molecular evidence for a natural diploid hybrid between <Emphasis Type="Italic">Miscanthus</Emphasis><Emphasis Type="Italic">sinensis</Emphasis> (Poaceae) and <Emphasis Type="Italic">M. sacchariflorus</Emphasis>

The hybrid origin of Miscanthus purpurascens has previously been proposed, primarily because of its intermediate morphology. In this study, phylogenies based on the DNA sequences from the internal transcribed spacer region of nuclear ribosomal DNA (nrDNA ITS), on the DNA sequences of the trnL intron and trnL-F intergenic spacer of chloroplast DNA, and on amplified fragment length polymorphism (AFLP) fingerprinting confirm that M. purpurascens originated through homoploid hybridization between M. sinensis and M. sacchariflorus. Two different types of ITS sequences were identified from almost all plants of M. purpurascens. One type was found to be closely related to M. sinensis and the other to M. sacchariflorus. Miscanthus purpurascens was found to possess many M. sinensis- and M. sacchariflorus-specific AFLP bands but no band specific to itself. Clustering with the Unweighted Pair Group Method with Arithmetic Mean and principal coordinate analysis based on the AFLP data also demonstrated that M. purpurascens is an approximate intermediate of the two species. In addition, M. purpurascens has the plastid genome of M. sinensis or M. sacchariflorus, suggesting that either species could be its maternal parent. All specimens of M. purpurascens and its coexisting parental species are identified as diploids (2n = 2x = 38). Possible mechanisms of natural hybridization, hybrid status, chloroplast DNA recombination, and evolutionary implications of this hybridization are also discussed.     

Molecular phylogeny of the genus <Emphasis Type="Italic">Bolusiella</Emphasis> (Orchidaceae,Angraecinae)

Recent molecular studies have suggested the monophyly of Bolusiella, a small orchid genus comprising five species and one subspecies from Continental Africa, but sampling has been limited. Using the species delimitation presented in the recent taxonomic revision of the genus, this study aimed to confirm the monophyly of Bolusiella and assess the interspecific relationships using a comprehensive sampling and various analytical methods. DNA sequences of one nuclear spacer region (ITS-1) and five plastid regions (matK, rps16, trnL–trnF, trnC–petN, and ycf1) from 20 specimens representing all five species of the genus were analyzed using static homology, dynamic homology, and Bayesian methods. The monophyly of both the genus Bolusiella and each of its five species was confirmed, corroborating the previously published taxonomic revision. The use of dynamic homology methods was not conclusive for this particular group. The results of the total evidence analysis (combining all six sequence regions) using the dynamic homology approach yielded a slightly different hypothesis regarding interspecific relationships (namely the exchange of B. talbotii and Bolusiella iridifolia as the earliest diverging lineage), probably because the nodes in question are supported by a small subset of conflicting characters, compared to the hypotheses resulting from the static homology and Bayesian methods, which are congruent with the results of previous studies.     

Inferring the potential of plastid DNA-based identification of derived ferns: a case study on the <Emphasis Type="Italic">Asplenium trichomanes</Emphasis> aggregate in Europe

The utility of three plastid DNA regions to identify fern species was explored with focus on the European representatives of the Asplenium trichomanes aggregate. The sampling included representatives of the three diploid and the four tetraploid taxa recognized in the European flora plus Macaronesia. Besides European samples, the compiled data set comprised specimens of a putative Hawaiian endemic and one species occurring in Southeast Asia. By combining the sequences of three non-coding plastid regions, 13 haplotypes were recovered of which four were found in more than one taxon. Evidences for four distinct diploid lineages were found that correspond to Asplenium anceps, A. inexpectans, A. trichomanes s.s., and A. tripteropus. The four tetraploids occurring in Europe shared haplotypes with A. inexpectans. Thus, DNA barcoding can successfully identify the diploids, but fail to separate the tetraploids from their diploid ancestors. As a consequence, barcoding analyses of ferns need to take into account the differences of ploidy level measured by evidence independent from the DNA barcode. Evidence for uneven accumulation of intra-species DNA variation was recovered by comparing all species. Furthermore, the study provided evidence that the current taxonomy of these ferns requires to be revised. The two European diploids form well-separated clades and need to be recognized as A. inexpectans and A. trichomanes s.s. To keep name consistency for all European tetraploids, a new name Asplenium jessenii is introduced to replace A. trichomanes subsp. hastatum.     

Unexpected presence of <Emphasis Type="Italic">Fagus orientalis</Emphasis> complex in Italy as inferred from 45,000-year-old DNA pollen samples from Venice lagoon

Background

Phylogeographic analyses on the Western Euroasiatic Fagus taxa (F. orientalis, F. sylvatica, F. taurica and F. moesiaca) is available, however, the subdivision of Fagus spp. is unresolved and there is no consensus on the phylogeny and on the identification (both with morphological than molecular markers) of Fagus Eurasiatic taxa.For the first time molecular analyses of ancient pollen, dated at least 45,000 years ago, were used in combination with the phylogeny analysis on current species, to identify the Fagus spp. present during the Last Interglacial period in Italy.In this work we aim at testing if the trn L-trn F chloroplast DNA (cpDNA) region, that has been previously proved efficient in discriminating different Quercus taxa, can be employed in distinguishing the Fagus species and in identifying the ancient pollen.

Results

86 populations from 4 Western Euroasistic taxa were sampled, and sequenced for the trn L-trn F region to verify the efficiency of this cpDNA region in identifying the Fagus spp.. Furthermore, Fagus crenata (2 populations), Fagus grandifolia (2 populations), Fagus japonica, Fagus hayatae, Quercus species and Castanea species were analysed to better resolve the phylogenetic inference.Our results show that this cpDNA region harbour some informative sites that allow to infer relationships among the species within the Fagaceae family. In particular, few specific and fixed mutations were able to discriminate and identify all the different Fagus species.Considering a short fragment of 176 base pairs within the trn L intron, 2 transversions were found able in distinguishing the F. orientalis complex taxa (F. orientalis, F. taurica and F. moesiaca) from the remaining Fagus spp. (F. sylvatica, F. japonica, F. hayataea, F. crenata and F. grandifolia). This permits to analyse this fragment also in ancient samples, where DNA is usually highly degraded.The sequences data indicate that the DNA recovered from ancient pollen belongs to the F. orientalis complex since it displays the informative sites characteristic of this complex.

Conclusion

The ancient DNA sequences demonstrate for the first time that, in contrast to current knowledge based on palynological and macrofossil data, the F. orientalis complex was already present during the Tyrrhenian period in what is now the Venice lagoon (Italy).This is a new and important insight considering that nowadays West Europe is not the natural area of Fagus orientalis complex, and up to now nobody has hypothesized the presence during the Last Interglacial period of F. orientalis complex in Italy.

     

Phylogeny of the Neotropical sages (<Emphasis Type="Italic">Salvia</Emphasis> subg. <Emphasis Type="Italic">Calosphace</Emphasis>; Lamiaceae) and insights into pollinator and area shifts

Salvia subg. Calosphace (Lamiaceae, Lamiales) is a highly diverse clade endemic to the New World. The phylogenetic relationships of Calosphace have been previously investigated using DNA sequences of nuclear ITS region and plastid psbA–trnH intergenic spacer, but the resulting trees lack resolution and support for many clades. The present paper reassesses the phylogenetic relationships of subgenus Calosphace, including a broader taxon sampling, with a special focus on representing previously unsampled sections, and using an additional plastid marker (trnL–trnF region). Our results show increased resolution and overall patterns of support, recovering ten main clades. Within core Calosphace, the most inclusive of these main clades, 17 new subclades were identified. Of the 42 sections for which more than one species was analysed, only 12 are monophyletic. Our biogeographical analysis identified at least twelve migrations to South America from Mexican and Central American lineages, in agreement with previous suggestions of multiple origins of South American Calosphace diversity. This analysis also confirmed a colonization of the Antilles by Andean lineages. The reconstruction of ancestral states of pollination syndromes showed multiple shifts to ornithophily from melittophily and one reversal to the latter.     

The complete chloroplast genome sequence of <Emphasis Type="Italic">Pseudoroegneria libanotica</Emphasis>, genomic features,and phylogenetic relationship with <Emphasis Type="Italic">Triticeae</Emphasis> species

Pseudoroegneria libanotica is an important herbage diploid species possessing the St genome. The St genome participates in the formation of nine perennial genera in Triticeae (Poaceae). The whole chloroplast (cp) genome of P. libanotica is 135 026 bp in length. The typical quadripartite structure consists of one large single copy of 80 634 bp, one small single copy of 12 766 bp and a pair of inverted regions (20 813 bp each). The cp genome contains 76 coding genes, four ribosomal RNA and 30 transfer RNA genes. Comparative sequence analysis suggested that: 1) the 737 bp deletion in the cp of P. libanotica was specific in Triticeae species and might transfer into its nuclear genome; 2) hot-spot regions, indels in intergenic regions and protein coding sequences mainly led to the length variation in Triticeae; 3) highly divergence regions combined with negative selection in rpl2, rps12, ccsA, rps8, ndhH, petD, ndhK, psbM, rps3, rps18, and ndhA were identified as effective molecular markers and could be considered in future phylogenetic studies of Triticeae species; and 4) ycf3 gene with rich cpSSRs was suitable for phylogeny analysis or could be used for DNA barcoding at low taxonomic levels. The cpSSRs distribution in the coding regions of diploid Triticeae species was shown for the first time and provided a valuable source for developing primers to study specific simple sequence repeat loci.     

New smut-specific primers for multilocus genotyping and phylogenetics of Ustilaginaceae

The Ustilaginomycotina, often collectively referred to as smut fungi, represent one of the three subphyla of the Basidiomycota. Smut fungi predominantly parasitize Angiosperms, are globally distributed, and contain several economically important pathogens. The most species-rich family of the smut fungi is the Ustilaginaceae. To investigate the molecular phylogeny of smut fungi, most studies rely on nrDNA loci, such as ITS and LSU. Protein coding genes, like rpb1, rpb2, TeF1a, atp6, and ß-tubulin, have been used in some studies. However, because of the huge diversity of smut fungi and the lack of dedicated primers, amplification of these loci has proven difficult for several groups. Thus, it was the aim of the current study to develop primers for new loci for the smut fungi with the focus on the largest family, the Ustilaginaceae. Here, the development and testing of new primers for nine loci based on protein-coding genes is reported (myosin, map, rpl3, tif2, ssc1, ß-tubulin, sdh1, rpl4A and atp2). A list of various primer combinations for the amplification of the new loci is given, with the corresponding PCR conditions and the best combinations for several genera of the Ustilaginaceae and some other Ustilaginomycetes. We hope that the primers presented in this study will be useful in overcoming the limitations of currently-used loci in terms of phylogenetic resolution, especially with respect to resolving species complexes and providing a better resolution of the higher-level phylogenetic relationships of smut fungi.     

Spatial patterns and intraspecific diversity of the glacial relict legume species <Emphasis Type="Italic">Vavilovia formosa</Emphasis> (Stev.) Fed. in Eurasia

Vavilovia formosa is one of five genera in tribe Fabeae, (Fabaceae, Leguminosae) with close phylogenetic relationships to Pisum. It grows in subalpine and alpine levels in Armenia, Azerbaijan, Georgia, Iran, Iraq, Lebanon, Russia and Turkey and is recognized as an endangered and protected plant. This study was conducted to reveal its intraspecific variability, as well as to predict the past, extant and future species distribution range. We analysed 51 accessions with common phylogenetic markers (trnF-trnL, trnS-trnG, matK, rbcL, psbA-trnH and ITS). These represent in total up to 2551 bp of chloroplast and 664 bp of nuclear sequences per sample. Two populations from Turkey and Armenia were analysed for genetic diversity by AFLP. Leaf morphometry was conducted on 1457 leaflets from 43 specimens. Extracted bioclimatic parameters were used for niche-modelling approach. Analysis of cpDNA revealed two haplotypes, 12 samples from Armenia, Daghestan, Nakhichevan and Iran belonged to H1 group, while 39 samples of all Turkish and part of Armenian were in H2 group. The mean intrapopulation diversity based on AFLP was low (H _E = 0.088) indicating limited outcrossing rate. A significantly positive correlation between geographical latitude and leaf area (\(\rho\) = 0.527, p < 0.05) was found. Niche modelling has shown temporal variation of predicted occurrence across the projected time periods. Vavilovia formosa has suffered a range reduction following climate warming after last glacial maximum, which classify this species as cold-adapted among the Fabeae species as well as a glacial relict.     

Diversity and evolution of leaf anatomical characters in Taxaceae <Emphasis Type="Italic">s.l.—</Emphasis>fluorescence microscopy reveals new delimitating characters

Taxaceae s.l. comprise six genera (including Cephalotaxus) and about 35 species; The present study aims to give new insights into the evolution of this family, especially into the phylogenetic position of Cephalotaxus. Moreover, only little is known about comparative leaf anatomy of this family and this study aims to expose and interpret the diversity and evolution of leaf anatomical characters and to assess their applicability to identify taxa at the generic and species level. A detailed phylogeny was reconstructed, using both maximum likelihood and Bayesian inference, with a combined dataset of four molecular markers from the plastid and nuclear genomes. Leaf sections from 132 specimens, representing 32 species and four varieties (fresh and herbarium material) were inspected, using fluorescence microscopy. Ancestral characters were reconstructed using Mesquite. The phylogenetic analyses provided full support for Cephalotaxus as sister group to Taxaceae s.str. Within the latter, two monophyletic tribes Taxeae (comprising Austrotaxus, Pseudotaxus, and Taxus) and Torreyeae (comprising Amentotaxus and Torreya) were fully supported. Fluorescence microscopy was shown to be very useful for identifying leaf tissues and their constitution. We were able to show that particularly sclerified tissues have highest potential for the discrimination of both freshly collected samples and rehydrated herbarium specimens at the generic and species level. A correlation between the presence of different sclereid types could be shown and sclereids were hypothesized to pose a primitive trait in the evolution of Taxaceae s.l. New identification keys were generated on the basis of leaf anatomical characters. The microscopic method presented here is applicable for further studies within gymnosperms and probably in angiosperms, as well.     

Conservation genetics and geographic patterns of genetic variation of endangered shrub <Emphasis Type="Italic">Ammopiptanthus</Emphasis> (Fabaceae) in northwestern China

Phylogeographic patterns of Ammopiptanthus in northwestern China were examined with internal transcribed spacer (ITS) and three chloroplast intergenic spacers (trnH–psbA, trnL–trnF, and trnS–trnG). Two ITS genotypes (a–b) and 8 chloroplast haplotypes (A–H) were detected. Both ITS genotypes and chloroplast lineages were split in two geographic regions: western Xinjiang and the Alxa Desert. This lineage split was also supported by AMOVA analysis and the Mantel test. AMOVA showed that 89.81 % of variance in Ammopiptanthus occurred between the two geographic regions, and correlation between genetic distances and geographical distances was significant (r = 0.757, p < 0.0001). All populations in western Xinjiang shared haplotype A with high frequency, and range expansion was strongly supported by negative Fu’s F_S value, and mismatch distribution analysis, whereas populations in the Alxa Desert had higher genetic diversity and structure. We speculate that the cold and dry climate during the early Quaternary fragmented habitats of the species, limiting gene flow between regions, and interglacial periods most likely led to the range expansion in western Xinjiang. The low genetic diversity of Ammopiptanthus indicate a significant extinction risk, and protective measures should be taken immediately.     

Polymorphism of Chloroplast DNA and Phylogeography of Green Alder (<Emphasis Type="Italic">Alnus alnobetula</Emphasis> (Ehrh.) K. Koch s. l.) in Asiatic Russia

This paper studies the phylogeography of Alnus alnobetula s. l. in the Asian part of the Russian range with the use of chloroplast DNA (cpDNA) sequences (intergenic spacers trnH-psbA, trnS-trnG, and psaA-trnS). Nine haplotypes were identified as a result of a combination of polymorphism of all sites in 241 plants in 20 populations of A. alnobetula. High interpopulation differentiation (G_ST = 0.914, N_ST = 0.928) and a significant phylogeographic structure (N_ST > G_ST, p < 0.05) were found. Reconstruction of genealogical relationships of green alder haplotypes revealed five highly divergent genetic lineages: one geographically widespread throughout Siberia and the Urals and several allopatric lines in the Far East of Russia, namely, the Chukotka Peninsula and the mouth of the Yana River, Magadan oblast, Primorye and the central part of Sakhalin Island, and the southern part of Sakhalin Island. Our data confirm that in the past the range of this species in the Far East was fragmented in several refugia that were isolated for a long time in various ice-free regions. This could have contributed to a high level of intraspecific polymorphism of green alder as a result of the divergence of endemic haplotypes, with almost non-overlapping distribution. However, the level of their molecular divergence does not correspond to the level of morphological differences.