The sources and quality of Iranian honey.

Iran is one of the largest honey-producing countries worldwide and is considered as an important source of honey for international markets. However, since Iran is not registered for honey export to Europe, the quality of Iranian honey remains unknown to European traders. As the first step in filling this gap, we analyzed 225 honey samples using palynology, sensory, nuclear magnetic resonance (NMR) and conventional physicochemical analyses as outlined by the European Union coordinated control plan. The results show that while various types of genuine unifloral honey can be harvested in Iran, 85% of collected samples were adulterated. Performing principal component analysis on physicochemical parameters reveals that feeding tablet sugar and syrup of C4 origin to bees during the foraging season is a common mode of fraud. Replacement of natural nectar with sugar syrup together with presence of intensive aftertaste from Taraxacum and Eryngium affect the taste of unifloral honeys produced in Iran.

Asymmetric contributions of seed and pollen to gene dispersal in the marsh orchid Dactylorhiza umbrosa in Asia Minor.

Orchids differ from other plants in their extremely small and partly air-filled seeds that can be transported long distances by wind. Seed dispersal in orchids is expected to contribute strongly to overall gene flow, and orchids generally express low levels of genetic differentiation between populations and low pollen to seed flow ratios. However, studies in orchids distributed in northern Europe have often found a poor geographic structuring of genetic variation. Here, we studied geographic differentiation in the marsh orchid Dactylorhiza umbrosa, which is widely distributed in upland regions from Asia Minor to Central Asia. These areas were less affected by Pleistocene ice ages than northern Europe and the orchid should have been able to survive the last ice age in local refugia. In the plastid genome, which is dispersed by seeds, populations at close distance were clearly divergent, but the differentiation still increased with geographic distance, and a significant phylogeographic structure had developed. In the nuclear genome, which is dispersed by both seeds and pollen, populations showed an even stronger correlation between genetic and geographic distance, but average levels of differentiation were lower than in the plastid genome, and no phylogeographic structure was evident. Combining plastid and nuclear data, we found that the ratio of pollen to seed dispersal (mp/ms) decreases with physical distance. Comparison with orchids that grow in parts of Europe that were glaciated during the last ice suggests that a balanced structure of genetic diversity develops only slowly in many terrestrial orchids, despite efficient seed dispersal.

Employing DNA metabarcoding to determine the geographical origin of honey.

Unfavourable climatic conditions force Iranian beekeepers to translocate over large distances in the course of the year. However, irrespective of the main place of production, the honey is always labeled with the name of the beekeepers' hometown, which leads consequently to mislabeled products. The present study investigates the capability of DNA metabarcoding to locate the geographical origin of honey. The molecular markers (ITS2 and rbcL) allowed identification of 926 plant species in studied samples. A comprehensive review of floristic reference books specified 34 key species that could be used to successfully determine the geographical origin in 91.4% of samples. These key species were usually present in honey with tiny amounts and thus, conventional palynology might not be able to detect them. The present investigation indicates that although ITS2 is able to detect more species than rbcL, utilizing a combination of both markers provides more robust evidence of geographical origin.

Phylogeny and species delimitation in Silene sect. Arenosae (Caryophyllaceae): a new section.

A putatively monophyletic group of annual Silene species is revised taxonomically and described as the new section S. sect. Arenosae. The species of this section were previously treated as a part of a widely circumscribed and polyphyletic S. sect. Rigidulae. Silene sect. Arenosae as circumscribed here consists of nine species. Members of the section show a predominantly E Mediterranean to SW Asian distribution pattern from Turkey southward to Egypt and eastward to Iran and Pakistan, although most of the species have a limited distribution range. The species of S. sect. Arenosae are characterized by narrowly lanceolate calyx teeth, which are often highly polymorphic, and lanceolate to oblanceolate (non-spathulate) basal leaves. The provided taxonomic revision is based on morphological characters and supported by phylogenetic analyses of two nuclear loci (nrITS and an intron of the RPB2 gene) and one chloroplast locus (the intron of the rps16 gene). The species descriptions are formalized using a novel implementation of the Prometheus Description Model.

Miocene climate change as a driving force for multiple origins of annual species in Astragalus (Fabaceae, Papilionoideae).

Astragalus, a highly diverse genus of flowering plants with its highest center of diversity in West Asia, is a classic example of rapid species-level radiation and adaptation to a diversity of habitats throughout the world. We examined the historical biogeography of Astragalus using molecular dating and ancestral area reconstruction to understand how past climate changes, geographical patterns and transition in life history have provoked diversification of Astragalus. Our results suggest that Astragalus probably originated during the middle Miocene in West Asia, underwent rapid diversification, subsequently and repeatedly expanded its range in the Mediterranean region, and later to North America through West Europe. This distribution range was also extended toward central and eastern Asia from the Middle Miocene to Pleistocene. Several climatic and geological processes during the Miocene-Pliocene may be implicated in the diversification of the major Astragalus clades. In particular, the annual lineages, which are important elements in the Mediterranean flora of Africa and Europe and in the deserts of southwest to central Asia, have arisen in response to progressing aridity from the late Miocene onwards (between 8.6 Ma and 2.98 Ma). Diversification rate analyses indicate three rapid and recent diversification events, one at c. 11 Ma in the clade that groups most of the Astragalus s.s. (all except the Ophiocarpus sister lineage), one at c. 5 Ma in the crown group of the Hypoglottis clade, including herbaceous annual and perennial species, and the most recent one at c. 3 Ma in the spiny cushion forming Astracantha clade. Our study highlights the complexity of processes and factors shaping diversifications in Astragalus; a complex interaction among climatic modifications providing opportunities for diversification and likely coincident with the evolution of key morphological and physiological adaptations.

Seed surface ultrastructure as an efficient tool for species delimitation in the Allium ampeloprasum L. alliance (Amaryllidaceae, Allioideae).

Seed surface ultrastructure shows a wide variation among different species of the genus Allium that could potentially provide valuable characters in inferring the relationships. In order to evaluate inter- and intraspecific variation of testa microsculpturing in the leek alliance, 33 seed samples of Allium representing its two subgenera, six sections, and 23 species were studied. Seed testa surface was analyzed using both SEM and Stereomicroscopy. Minor differences in testa cell arrangement were observed between members of sect. Allium and sect. Avulsea (both of subg. Allium), but major contrasts were discerned between them and members of subg. Cepa. Significant similarity was distinguished in anticlinal walls of the members of sect. Allium and sect. Avulsea: they were variously undulated (U-, to ±Ω-like). Testa cells in members of subg. Cepa, and some of subg. Allium (sect. Caerulea, sect. Codonoprasum, and sect. Longivaginata) showed ribbed anticlinal walls suggesting a close relationship between them. The periclinal walls were concave, flat to convex with testa cells densely granulose at margins in sect. Caerulea; flat to convex, irregularly granulose-verrucose in sect. Codonoprasum; concave, densely granulose at margins and center in sect. Longivaginata; and convex, densely irregularly granulose, sometimes centrally verrucose in subg. Cepa. Irregularly polygonal cells were present in most species, but elongated polygonal cells in two species, A. iranicum and A. wendelboi (sect. Allium). We found significant micromorphological similarity in seed surface features between A. abbasii and A. fuscoviolaceum; A. esfandiarii and A. atroviolaceum; and A. longipapillatum and A. rotundum, corroborating close relationships.

Evaluation of storage time effect on saffron chemical profile using gas chromatography and spectrophotometry techniques coupled with chemometrics.

Saffron quality is commonly determined by three parameters: color, aroma, and taste. Several factors including harvesting and post-harvesting conditions, affect these parameters. In this study, the effect of storage time on saffron quality was evaluated. At first, the relative concentration of the saffron secondary metabolites in freshly dried and 2 years stored saffron samples prepared with ISO 3632 and UA-DLLME methods and then measured using UV-Vis and GC-FID techniques. In order to find saffron storage time biomarkers, the obtained data were subjected to several data analysis steps including data preprocessing, principal component analysis (PCA), partial least square discriminant analysis (PLS-DA) and variable selection methods. Based on the obtained main biomarkers and proposed molecule mechanism, it can be concluded that during the storage periods, the intensity of saffron color reduces, while its aroma increases, reflecting a negative correlation between them. Freshly dried samples have a higher level of the crocins as coloring agents, ß-isophorone, 4-hydroxy-3,5,5-trimethylcyclohex-2-enone and picrocrocin, while the stored samples were more abundant by safranal as the main saffron aroma agent.

An image analysis of TLC patterns for quality control of saffron based on soil salinity effect: A strategy for data (pre)-processing.

Quality of saffron, a valuable food additive, could considerably affect the consumers' health. In this work, a novel preprocessing strategy for image analysis of saffron thin layer chromatographic (TLC) patterns was introduced. This includes performing a series of image pre-processing techniques on TLC images such as compression, inversion, elimination of general baseline (using asymmetric least squares (AsLS)), removing spots shift and concavity (by correlation optimization warping (COW)), and finally conversion to RGB chromatograms. Subsequently, an unsupervised multivariate data analysis including principal component analysis (PCA) and k-means clustering was utilized to investigate the soil salinity effect, as a cultivation parameter, on saffron TLC patterns. This method was used as a rapid and simple technique to obtain the chemical fingerprints of saffron TLC images. Finally, the separated TLC spots were chemically identified using high-performance liquid chromatography-diode array detection (HPLC-DAD). Accordingly, the saffron quality from different areas of Iran was evaluated and classified.

DNA barcoding of tuberous Orchidoideae: a resource for identification of orchids used in Salep.

Tubers of terrestrial orchids are harvested and traded from the eastern Mediterranean to the Caspian Sea for the traditional product Salep. Overexploitation of wild populations and increased middle-class prosperity have escalated prices for Salep, causing overharvesting, depletion of native populations and providing an incentive to expand harvesting to untapped areas in Iran. Limited morphological distinctiveness among traded Salep tubers renders species identification impossible, making it difficult to establish which species are targeted and affected the most. In this study, a reference database of 490 nrITS, trnL-F spacer and matK sequences of 133 taxa was used to identify 150 individual tubers from 31 batches purchased in 12 cities in Iran to assess species diversity in commerce. The sequence reference database consisted of 211 nrITS, 158 trnL-F and 121 matK sequences, including 238 new sequences from collections made for this study. The markers enabled unambiguous species identification with tree-based methods for nrITS in 67% of the tested tubers, 58% for trnL-F and 59% for matK. Species in the genera Orchis (34%), Anacamptis (27%) and Dactylorhiza (19%) were the most common in Salep. Our study shows that all tuberous orchid species in this area are threatened by this trade, and further stresses the urgency of controlling illegal harvesting and cross-border trade of Salep tubers.

Assembled Plastid and Mitochondrial Genomes, as well as Nuclear Genes, Place the Parasite Family Cynomoriaceae in the Saxifragales.

Cynomoriaceae, one of the last unplaced families of flowering plants, comprise one or two species or subspecies of root parasites that occur from the Mediterranean to the Gobi Desert. Using Illumina sequencing, we assembled the mitochondrial and plastid genomes as well as some nuclear genes of a Cynomorium specimen from Italy. Selected genes were also obtained by Sanger sequencing from individuals collected in China and Iran, resulting in matrices of 33 mitochondrial, 6 nuclear, and 14 plastid genes and rDNAs enlarged to include a representative angiosperm taxon sampling based on data available in GenBank. We also compiled a new geographic map to discern possible discontinuities in the parasites' occurrence. Cynomorium has large genomes of 13.70-13.61 (Italy) to 13.95-13.76 pg (China). Its mitochondrial genome consists of up to 49 circular subgenomes and has an overall gene content similar to that of photosynthetic angiosperms, while its plastome retains only 27 of the normally 116 genes. Nuclear, plastid and mitochondrial phylogenies place Cynomoriaceae in Saxifragales, and we found evidence for several horizontal gene transfers from different hosts, as well as intracellular gene transfers.

Molecular phylogeny of tribe Stachydeae (Lamiaceae subfamily Lamioideae).

Although tribe Stachydeae (Lamiaceae) is considered monophyletic, relationships within the tribe are still poorly understood. The complexity of Stachydeae includes paraphyletic genera, considerable morphological plasticity, a range of ploidy levels, and presumably frequent natural hybridization. We performed parsimony and Bayesian phylogenetic analyses of nuclear (ribosomal ITS) and plastid (trnL intron, trnL-trnF spacer, rps16 intron) DNA sequence data from a taxonomically and geographically broad sampling of the tribe to identify major evolutionary lineages and to test taxonomic hypotheses within this largest of all lamioid tribes. We included 143 accessions corresponding to 121 species, representing both Old and New World species, and all 12 recognized genera of tribe Stachydeae. Both nuclear and plastid data corroborate monophyly of the tribe, with Melittis as sister to all remaining Stachydeae. For the latter well-supported clade, we suggest the phylogenetic name Eurystachys. Within Eurystachys, although monophyly is supported by both nuclear and plastid data for several named and unnamed groups, the majority of recognized taxa appear to be para- or polyphyletic. The taxon compositions of most subclades are congruent between the plastid and nuclear tree topologies, whereas their relative phylogenetic placements are often not. This level of plastid-nuclear incongruence suggests considerable impact of hybridization in the evolution of Stachydeae.

Regional and local patterns of ectomycorrhizal fungal diversity and community structure along an altitudinal gradient in the Hyrcanian forests of northern Iran.

• Altitudinal gradients strongly affect the diversity of plants and animals, yet little is known about the altitudinal effects on the distribution of microorganisms, including ectomycorrhizal fungi. • By combining morphological and molecular identification methods, we addressed the relative effects of altitude, temperature, precipitation, host community and soil nutrient concentrations on species richness and community composition of ectomycorrhizal fungi in one of the last remaining temperate old-growth forests in Eurasia. • Molecular analyses revealed 367 species of ectomycorrhizal fungi along three altitudinal transects. Species richness declined monotonically with increasing altitude. Host species and altitude were the main drivers of the ectomycorrhizal fungal community composition at both the local and regional scales. The mean annual temperature and precipitation were strongly correlated with altitude and accounted for the observed patterns of richness and community. • The decline of ectomycorrhizal fungal richness with increasing altitude is consistent with the general altitudinal richness patterns of macroorganisms. Low environmental energy reduces the competitive ability of rare species and thus has a negative effect on the richness of ectomycorrhizal fungi. Because of multicollinearity with altitude, the direct effects of climatic variables and their seasonality warrant further investigation at the regional and continental scales.

Phylogeny and character evolution in Medicago (Leguminosae): Evidence from analyses of plastid trnK/matK and nuclear GA3ox1 sequences.

PREMISE OF THE STUDY: The genus Medicago, with about 87 species, includes the model legume species M. truncatula, and a number of important forage species such as M. sativa (alfalfa), M. scutellata (snail medic), and M. lupulina (black medic). Relationships within the genus are not yet sufficiently resolved, contributing to difficulty in understanding the evolution of a number of distinguishing characteristics such as aneuploidy and polyploidy, life history, structure of cotyledons, and number of seeds per fruit. • METHODS: Phylogenetic relationships of 70-73 species of Medicago and its sister genus Trigonella (including Melilotus) were reconstructed from nucleotide sequences of the plastid trnK/matK region and the nuclear-encoded GA3ox1 gene (gibberellin 3-ß-hydroxylase) using maximum parsimony and Bayesian inference methods. • KEY RESULTS: Our results support certain currently recognized taxonomic groups, e.g., sect. Medicago (with M. sativa) and sect. Buceras. However, other strongly supported clades-the "reduced subsection Leptospireae clade" that includes M. lupulina, the "polymorpha clade" that includes M. murex and M. polymorpha and the "subsection Pachyspireae clade" that includes M. truncatula-each of which includes species presently in different subsections of sect. Spirocarpos, contradict the current classification. • CONCLUSIONS: These results support the hypothesis that some characters considered important in existing taxonomies, for example, single-seeded fruits that have arisen more than once in both Medicago and Trigonella, are indeed homoplastic. Others, such as the 2n = 14 chromosome number, have also arisen independently within the genus. In addition, we demonstrate support for the utility of GA3ox1 sequences for phylogenetic analysis among and within closely related genera of legumes.