Genotyping-by-sequencing reveals range expansion of Adonis vernalis (Ranunculaceae) from Southeastern Europe into the zonal Euro-Siberian steppe.

The Euro-Siberian steppe flora consists of warm- and cold-adapted species, which may have responded differently to Pleistocene glacials and interglacials. Genotyping-by-sequencing individuals from across the distribution range of the pheasant's eye (Adonis vernalis), we aimed to gain insight into steppe florogenesis based on the species' evolutionary history. Although the primary area of origin of the species group comprising A. vernalis, A. villosa and A. volgensis is in Asia, our results indicate that recent populations of A. vernalis are not of Asian origin but evolved in the southern part of Europe during the Pleistocene, with Spanish populations clearly genetically distinct from the Southeastern European populations. We inferred that A. vernalis migrated eastwards from the sub-Mediterranean forest-steppes of Southeastern Europe into the continental forest-steppe zone. Eastern European populations had the highest private allelic richness, indicating long-term large population sizes in this region. As a thermophilic species, A. vernalis seems unlikely to have survived in the cold deserts of the Last Glacial Maximum in Western Siberia, so this region was likely (re)colonized postglacially. Overall, our results reinforce the importance of identifying the area of origin and the corresponding ecological requirements of steppe plants in order to understand the composition of today's steppe flora.

Allium pallasii and A. caricifolium-Surprisingly Diverse Old Steppe Species, Showing a Clear Geographical Barrier in the Area of Lake Zaysan.

Polymorph Allium pallasii s.l. from monotypic A. sect. Pallasia was studied using a wide spectrum of methods and divided into two clearly morphologically, geographically, cytologically and genetically isolated species: A. pallasii s. str.-North-East Kazakhstan, Western Siberia, and the Altai Mountains; A. caricifolium-Kyrgyzstan, Northwest China, South-East Kazakhstan until Zaysan Lake in the east. Despite serious genetic differences, both species are sisters and are related to species of the A. sect. Codonoprasum (Subg. Allium). Allium caricifolium differs from A. pallasii s. str. by taller stems, dense inflorescence, and with filaments longer than perianth. The possible phylogenetic reasons for the separation of these species are discussed. A nomenclature analysis of synonyms was carried out.

Biodiversity of Duckweed (Lemnaceae) in Water Reservoirs of Ukraine and China Assessed by Chloroplast DNA Barcoding.

Monitoring and characterizing species biodiversity is essential for germplasm preservation, academic studies, and various practical applications. Duckweeds represent a group of tiny aquatic plants that include 36 species divided into 5 genera within the Lemnaceae family. They are an important part of aquatic ecosystems worldwide, often covering large portions of the water reservoirs they inhabit, and have many potential applications, including in bioremediation, biofuels, and biomanufacturing. Here, we evaluated the biodiversity of duckweeds in Ukraine and Eastern China by characterizing specimens using the two-barcode protocol with the chloroplast atpH-atpF and psbK-psbI spacer sequences. In total, 69 Chinese and Ukrainian duckweed specimens were sequenced. The sequences were compared against sequences in the NCBI database using BLAST. We identified six species from China (Spirodela polyrhiza, Landoltia punctata, Lemna aequinoctialis, Lemna minor, Lemna turionifera, and Wolffia globosa) and six from Ukraine (S. polyrhiza, Lemna gibba, Lemna minor, Lemna trisulca, Lemna turionifera, and Wolffia arrhiza). The most common duckweed species in the samples from Ukraine were Le. minor and S. polyrhiza, accounting for 17 and 15 out of 40 specimens, respectively. The most common duckweed species in the samples from China was S. polyrhiza, accounting for 15 out of 29 specimens. La. punctata and Le. aequinoctialis were also common in China, accounting for five and four specimens, respectively. According to both atpH-atpF and psbK-psbI barcode analyses, the species identified as Le. aequinoctialis does not form a uniform taxon similar to other duckweed species, and therefore the phylogenetic status of this species requires further clarification. By monitoring duckweeds using chloroplast DNA sequencing, we not only precisely identified local species and ecotypes, but also provided background for further exploration of native varieties with diverse genetic backgrounds. These data could be useful for future conservation, breeding, and biotechnological applications.

Allium sulaimanicum: A new Allium species and section from Pakistan.

A new species, Allium sulaimanicum, is described from northern Balochistan and southern Khyber Pakhtunkhwa in Pakistan based on morphological, molecular, and cytological studies. The new species is characterised by long runner-like cylindrical rhizomes of adult plants, cylindrical bulbs, linear leaves with minute soft hairs along veins, campanulate perigonium, and white to creamy white, ovate to elliptical, 4.5-5-mm-long acute tepals, with brownish to purplish nerves, stamens as long as to slightly longer than tepals, yellow to brick red anthers, hexagonal ovary, and white and papillate/warty along angles. The presence of long herbaceous rhizomes indicated serious isolation of the new species; hence, a new section Sulaimanicum is proposed to accommodate the new species. The new species is diploid with a chromosome number of 2n = 16. Detailed morphological description, illustrations, phylogenetic analyses based on sequences of plastid spacers (rpl32-trnL (UAG) and trnQ-rps16) and nuclear ITS, karyotype features, and a distribution map of the new species are provided.

The phylogeographic history of Krascheninnikovia reflects the development of dry steppes and semi-deserts in Eurasia.

Constituting one of Earth's major biomes, steppes are characterised by naturally treeless extra-tropical vegetation. The formation of the Eurasian steppe belt, the largest steppe region in the world, began in Central Asia during the Neogene. In the glacial stages of the Pleistocene, steppe displaced forest vegetation, which in turn recolonised the area during the warmer interglacial periods, thus affecting the distribution of plants adapted to these habitats. Krascheninnikovia ceratoides (Chenopodiaceae) is a plant characteristic of dry steppe and semi-desert formations. Earlier studies showed that the ancestor of this autochthonous steppe element originated in Central Asia during the Miocene/Pliocene, i.e., in the same region and at the same time as the first appearance of steppe vegetation. However, as the extant lineages of Krascheninnikovia ceratoides diversified only 2.2 ± 0.9 Mya, it may represent a modern element of current dry steppe and semi-desert formations, rather than a component of the first steppe precursors of the Miocene. As such, it may have capitalised on the climatic conditions of the cold stages of the Quaternary to expand its range and colonise suitable habitats outside of its area of origin. To test this hypothesis, phylogeographic methods were applied to high-resolution genotyping-by-sequencing data. Our results indicate that Krascheninnikovia originated in western Central Asia and the Russian Altai, then spread to Europe in the West, and reached North America in the East. The populations of eastern Central Asia and North America belong to the same clade and are genetically clearly distinct from the Euro-Siberian populations. Among the populations west of the Altai Mountains, the European populations are genetically distinct from all others, which could be the result of the separation of populations east and west of the Urals caused by the Pleistocene transgressions of the Caspian Sea.

Seed Morphology of Allium L. (Amaryllidaceae) from Central Asian Countries and Its Taxonomic Implications.

We studied seed macro- and micro-morphological characteristics of 48 Allium species (51 accessions) belonging to 24 sections and 7 subgenera. Our taxonomic sampling focused on the central Asian regions of Uzbekistan, Kyrgyzstan, and Mongolia. The seed length ranged between 1.74 ± 0.16-4.47 ± 0.43 mm and width ranged between 1.06 ± 0.08-3.44 ± 0.23 mm, showing various shapes. The irregular and elongated polygonal testa cells occurred in all investigated species. Seed testa sculptures showed high variation in their anticlinal walls associated with different shapes: straight to with U-, S- or Omega-type undulations among the species. The moderately flat to convex periclinal walls with various sized verrucae or granules were found in all investigated taxa. Based on our research, we conclude that seed characteristics such as size, shape, and the seed testa features show their significant variability, revealing key characteristics to support taxonomic relationships and major clades recovered in the molecular phylogeny of the genus Allium. Especially, the anticlinal wall characteristics were highly variable and decisive at the both section and species levels. In addition, widely varied shapes and sizes of the seeds were remarkably effective to distinguish Allium species.

Genetic diversity of Helosciadium repens (Jacq.) W.D.J. Koch (Apiaceae) in Germany, a Crop Wild Relative of celery.

Helosciadium repens (Jacq.) W.D.J. Koch is threatened by genetic erosion. It is a Crop Wild Relative (CWR) of celery and celeriac and a potentially valuable genetic resource for plant breeding. The objective of this study was the analysis of distribution of genetic diversity with a set of selected populations in Germany. The results of the genetic analysis and data obtained during the site visits were used to identify a subset which was chosen to best represent the genetic diversity of H. repens in Germany. The chance of long-term conservation by securing the identified populations in genetic reserves is distinctly possible.Seven hundred and fifteen individuals from 27 sites were assessed using six simple sequence repeat markers. Discriminant analysis of principal components was used to identify six clusters of genetically similar individuals. The complementary compositional genetic differentiation Δj was calculated to designate a subset of populations chosen to best represent the overall genetic diversity. Entry 18R (Δ18R = 0.2498) represented its pooled remainder the best, while entry 22R (Δ22R = 0.4902) differed the most from its complement.Based on the results of the genetic analysis and information regarding the current conservation status, 14 most appropriate wild populations for potential genetic reserve were identified. The used markers display a low level of genetic variation between the analyzed populations, and a split between Northern and Southern populations was observed.CWR species are essential genetic resources for plant breeding and food security. However, 11.5% of the European CWRs are threatened. Therefore, it is of utmost importance to determine their genetic compositions. These insights will provide the fundamental basis for making crucial decisions concerning future conservation strategies for H. repens.

Ecotypes or phenotypic plasticity-The aquatic and terrestrial forms of Helosciadium repens (Apiaceae).

Morphological and ecological differences of two forms of Helosciadium repens are known and described in the literature: aquatic and terrestrial. However, their taxonomic status is currently unknown. The question whether they are genotypically adapted to specific environmental conditions or are those differences a result of phenotypic plasticity is addressed in this study.SSR and ISSR data were used to uncover genotypic differences. Data from drought stress experiments (system water content and relative water content of leaves) were used to evaluate the response to water as an environmental factor. The stomatal index of both forms grown under different water treatments was analyzed.The principal component analysis of the ISSR data revealed no clustering that would correspond with ecotypes. The diversity parameters of the SSR data showed no significant differences. The aquatic populations showed a tendency toward heterozygosity, while the terrestrial ones showed a bias toward homozygosity. Both forms responded similarly to the changes in water availability, with newly produced leaves after drought stress that were better adapted to repeated drought stress. Stomatal indices were higher in plants from aquatic habitats, but these differences disappeared when the plants were grown in soil.The observed responses indicate that the differences between forms are due to phenotypic plasticity.

Phylogeny of Allium L. subgenus Anguinum (G. Don. ex W.D.J. Koch) N. Friesen (Amaryllidaceae).

The disjunct distribution of the subgenus Anguinum of the genus Allium makes it a good candidate to test models of Northern hemisphere biogeography. Here we conduct phylogenetic analysis with the nuclear marker ITS and three different chloroplast markers (rps16 intron, rbcL-atpB spacer, rpl32-trnL spacer). Divergence time estimations (Beast) relying on published ITS substitution rates and ancestral range reconstructions were calculated to elucidate the biogeographical history of the subgenus. Additionally we compiled distribution maps for all species with data taken from the literature, herbariums and data from field observations. The main radiation of the subgenus took place in the last one million years and is still going on. They have their origins in the mid Miocene in East Asia and were highly influenced by the climate fluctuations in the Pliocene/Pleistocene period. Conflicting tree topologies between nuclear and cpDNA markers of Allium tricoccum Solander indicate that the species is of hybridogenous origin. Cloning the ITS sequence revealed the parental copies and confirmed our conclusion. One originated from the Eurasian and the other from the East Asian clade. We were able to show that it reached North America most likely via the Beringia around 2.5mya (95% HPD of 0.35-5.26mya). Our data suggest that Allium victorialis L. is only distributed in mountain pastures in Europe as it forms a well-supported clade in the ITS tree. In the analysis of the molecular markers we found two distinct types of Allium ochotense Prokh. and we suggest splitting the species based on Prokhanov's (1930) proposal. Taxonomical remarks and an identification key to all species of the subgenus Anguinum is provided.

'Missing link' species Capsella orientalis and Capsella thracica elucidate evolution of model plant genus Capsella (Brassicaceae).

To elucidate the evolutionary history of the genus Capsella, we included the hitherto poorly known species C. orientalis and C. thracica into our studies together with C. grandiflora, C. rubella and C. bursa-pastoris. We sequenced the ITS and four loci of noncoding cpDNA regions (trnL - F, rps16, trnH -psbA and trnQ -rps16). Sequence data were evaluated with parsimony and Bayesian analyses. Divergence time estimates were carried out with the software package BEAST. We also performed isozyme, cytological, morphological and biogeographic studies. Capsella orientalis (self-compatible, SC; 2n = 16) forms a clade (eastern lineage) with C. bursa-pastoris (SC; 2n = 32), which is a sister clade (western lineage) to C. grandiflora (self-incompatible, SI; 2n = 16) and C. rubella (SC; 2n = 16). Capsella bursa-pastoris is an autopolyploid species of multiple origin, whereas the Bulgarian endemic C. thracica (SC; 2n = 32) is allopolyploid and emerged from interspecific hybridization between C. bursa-pastoris and C. grandiflora. The common ancestor of the two lineages was diploid and SI, and its distribution ranged from eastern Europe to central Asia, predominantly confined to steppe-like habitats. Biogeographic dynamics during the Pleistocene caused geographic and genetic subdivisions within the common ancestor giving rise to the two extant lineages.

Molecular evidence for bicontinental hybridogenous genomic constitution in Lepidium sensu stricto (Brassicaceae) species from Australia and New Zealand.

Lepidium sensu stricto (s.s.) (Brassicaceae) (ca. 150 species) is distributed worldwide with endemic species on every continent. It is represented in Australia and New Zealand by 19 and seven native species, respectively. In the present study we used a nuclear ribosomal internal transcribed spacer (ITS) phylogeny in comparison with a cpDNA phylogeny to unravel the origin of Australian/New Zealand species. Although phylogenetic relationships within Lepidium s.s. were not fully resolved, the cpDNA data were in agreement with a Californian origin of Lepidium species from Australia/New Zealand. Strongly conflicting signals between the cp- and nuclear DNA phylogenetic analysis clearly indicated hybridogenous genomic constitution of Australian Lepidium s.s. species: All 18 studied Australian/New Zealand Lepidium s.s. species examined shared a Californian cpDNA type. While eleven Australian/New Zealand species appeared to harbor a Californian ITS type, a group of seven species shared a South African ITS type. This pattern is most likely explained by two trans-oceanic dispersals of Lepidium from California and Africa to Australia/New Zealand and subsequent hybridization followed by homogenization of the ribosomal DNA either to the Californian or South African ITS type in the two different lineages. Calibration of our molecular trees indicates a Pliocene/Pleistocene origin of Lepidium in Australia/New Zealand. Low levels of cpDNA and ITS sequence divergence and unresolved topologies within Australian/New Zealand species suggest a rapid and recent radiation of Lepidium after the hybridization event. This coincides with dramatic climatic changes in that geological epoch shaping the composition of the vegetation.

Molecular cytogenetic analysis of Podocarpus and comparison with other gymnosperm species.

DNA sequences have been mapped to the chromosomes of Podocarpus species from New Zealand and Australia by fluorescent in situ hybridization. Unlike other conifers, these species show only one pair of major sites of 45S rDNA genes, and two additional minor sites were seen in the Australian P. lawrencei. Unusually, 45S sequences collocalize to the same chromosomal region as the 5S rDNA. The telomere probe (TTTAGGG)n hybridizes to the ends of all chromosomes as well as to a large number of small sites distributed along the length of all chromosomes. Two other simple sequence repeats, (AAC)5 and (GATA)4, show a diffuse pattern of hybridization sites distributed along chromosomes. Southern blots using a variety of probes obtained from the reverse transcriptase of retroelements (gypsy, copia and LINE) from P. totara, P. nivalis and Dacrycarpus dacrydioides show that these retroelements are abundant and widespread in Podocarpaceae and also in others conifers. Some retroelements such as copia pPonty3 and gypsy pPot1li are more abundant in the genome of Picea abies and Ginkgo biloba than in the species from which they were amplified.