Evolutionary history of an Irano-Turanian cushion-forming legume (Onobrychis cornuta).

The Irano-Turanian region is one of the largest floristic regions in the world and harbors a high percentage of endemics, including cushion-like and dwarf-shrubby taxa. Onobrychis cornuta is an important cushion-forming element of the subalpine/alpine flora of the Irano-Turanian floristic region. To specify the genetic diversity among the populations of this species (including individuals of O. elymaitica), we employed nrDNA ITS and two noncoding regions of plastid DNA (rpl32-trnL(UAG) and trnT(UGU)-trnL(UAA)). The most striking feature of O. cornuta assemblages was the unexpectedly high nucleotide diversity in both the nDNA and cpDNA dataset. In the analyses of nuclear and plastid regions, 25 ribotypes and 42 haplotypes were found among 77 and 59 accessions, respectively, from Iran, Turkey, and Afghanistan. Network analysis of the datasets demonstrated geographic differentiation within the species. Phylogenetic analyses of all dataset retrieved O. cornuta as a non-monophyletic species due to the inclusion of O. elymaitica, comprising four distinct lineages. In addition, our analyses showed cytonuclear discordance between both nuclear and plastid topologies regarding the position of some O. cornuta individuals. The underlying causes of this inconsistency remain unclear. However, we speculate that chloroplast capture, incomplete lineage sorting, and introgression were the main reasons for this event. Furthermore, molecular dating analysis indicated that O. cornuta originated in the early Pliocene (around 4.8 Mya) and started to diversify throughout the Pliocene and in particular the Pleistocene. Moreover, O. elymaitica was reduced to a subspecific rank within the species.

Characterization and comparative analysis of the complete plastid genomes of four Astragalus species.

Astragalus is the largest flowering plant genus. We assembled the plastid genomes of four Astragalus species (Astragalus iranicus, A. macropelmatus, A. mesoleios, A. odoratus) using next-generation sequencing and analyzed their plastomes including genome organization, codon usage, nucleotide diversity, prediction of RNA editing and etc. The total length of the newly sequenced Astragalus plastomes ranged from 121,050 bp to 123,622 bp, with 110 genes comprising 76 protein-coding genes, 30 transfer RNA (tRNA) genes and four ribosome RNA (rRNA) genes. Comparative analysis of the chloroplast genomes of Astragalus revealed several hypervariable regions comprising three non-coding sites (trnQ(UUG)-accD, rps7 -trnV(GAC) and trnR(ACG)-trnN(GUU)) and four protein-coding genes (ycf1, ycf2, accD and clpP), which have potential as molecular markers. Positive selection signatures were found in five genes in Astragalus species including rps11, rps15, accD, clpP and ycf1. The newly sequenced species, A. macropelmatus, has an approximately 13-kb inversion in IR region. Phylogenetic analysis based on 75 protein-coding gene sequences confirmed that Astragalus form a monophyletic clade within the tribe Galegeae and Oxytropis is sister group to the Coluteoid clade. The results of this study may helpful in elucidating the chloroplast genome structure, understanding the evolutionary dynamics at genus Astragalus and IRLC levels and investigating the phylogenetic relationships. Moreover, the newly plastid genomes sequenced have been increased the plastome data resources on Astragalus that can be useful in further phylogenomic studies.

Characterizing nrDNA ITS1, 5.8S and ITS2 secondary structures and their phylogenetic utility in the legume tribe Hedysareae with special reference to Hedysarum.

This is the first study to systematically evaluate rRNA secondary structures of Hedysareae with an emphasis on Hedysarum. ITS2 and 5.8S regions of the genus shared a common secondary structure with a four-fingered central loop, whereas ITS1 possessed five distinct structures. The secondary structural features of the two regions provided advantageous data for clades, species groups, and closely related species. Hemi-CBCs were mostly observed in the reconstruction of species groups, and Nsts, mostly between closely related species. The investigations showed that ITS1 varied more than ITS2 in length, GC content, and most of the diversity indices within the tribe. Maximum likelihood analyses of the synchronized sequence-structure tree of ITS1 were performed. The accuracy and phylogenetic signals of ITS1 were higher than ITS2. The similar GC content, and no CBC, in both spacers, fortified the close relationship of CEGO and H. sections Stracheya and Hedysarum clades in the synchronized sequence-structure tree topology of ITS1. In both regions, no inter-generic CBCs were detected inside the CEGO clade and the inter-sectional level of Hedysarum. But, in the ITS2 region, a CBC was detected between H. section Multicaulia, and Taverniera versus H. sections Hedysarum, and Stracheya. The lowest inter-sectional genetic distance and structural features were found between H. sect. Hedysarum and H. sect. Stracheya clades in the ITS2 region.

A phylogenetic analysis of Bromus (Poaceae: Pooideae: Bromeae) based on nuclear ribosomal and plastid data, with a focus on Bromus sect. Bromus.

To investigate phylogenetic relationships among and within major lineages of Bromus, with focus on Bromus sect. Bromus, we analyzed DNA sequences from two nuclear ribosomal (ITS, ETS) and two plastid (rpl32-trnLUAG , matK) regions. We sampled 103 ingroup accessions representing 26 taxa of B. section Bromus and 15 species of other Bromus sections. Our analyses confirm the monophyly of Bromus s.l. and identify incongruence between nuclear ribosomal and plastid data partitions for relationships within and among major Bromus lineages. Results support classification of B. pumilio and B. gracillimus within B. sect. Boissiera and B. sect. Nevskiella, respectively. These species are sister groups and are closely related to B. densus (B. sect. Mexibromus) in nrDNA trees and Bromus sect. Ceratochloa in plastid trees. Bromus sect. Bromopsis is paraphyletic. In nrDNA trees, species of Bromus sects. Bromopsis, Ceratochloa, Neobromus, and Genea plus B. rechingeri of B. sect. Bromus form a clade, in which B. tomentellus is sister to a B. sect. Genea-B. rechingeri clade. In the plastid trees, by contrast, B. sect. Bromopsis species except B. tomentosus form a clade, and B. tomentosus is sister to a clade comprising B. sect. Bromus and B. sect. Genea species. Affinities of B. gedrosianus, B. pulchellus, and B. rechingeri (members of the B. pectinatus complex), as well as B. oxyodon and B. sewerzowii, are discordant between nrDNA and plastid trees. We infer these species may have obtained their plastomes via chloroplast capture from species of B. sect. Bromus and B. sect. Genea. Within B. sect. Bromus, B. alopecuros subsp. caroli-henrici, a clade comprising B. hordeaceus and B. interruptus, and B. scoparius are successive sister groups to the rest of the section in the nrDNA phylogeny. Most relationships among the remaining species of B. sect. Bromus are unresolved in the nrDNA and plastid trees. Given these results, we infer that most B. sect. Bromus species likely diversified relatively recently. None of the subdivisional taxa proposed for Bromus sect. Bromus over the last century correspond to natural groups identified in our phylogenetic analyses except for a group including B. hordeaceus and B. interruptus.

The complete chloroplast genome of Onobrychis gaubae (Fabaceae-Papilionoideae): comparative analysis with related IR-lacking clade species.

BACKGROUND: Plastome (Plastid genome) sequences provide valuable markers for surveying evolutionary relationships and population genetics of plant species. Papilionoideae (papilionoids) has different nucleotide and structural variations in plastomes, which makes it an ideal model for genome evolution studies. Therefore, by sequencing the complete chloroplast genome of Onobrychis gaubae in this study, the characteristics and evolutionary patterns of plastome variations in IR-loss clade were compared. RESULTS: In the present study, the complete plastid genome of O. gaubae, endemic to Iran, was sequenced using Illumina paired-end sequencing and was compared with previously known genomes of the IRLC species of legumes. The O. gaubae plastid genome was 122,688 bp in length and included a large single-copy (LSC) region of 81,486 bp, a small single-copy (SSC) region of 13,805 bp and one copy of the inverted repeat (IRb) of 29,100 bp. The genome encoded 110 genes, including 76 protein-coding genes, 30 transfer RNA (tRNA) genes and four ribosome RNA (rRNA) genes and possessed 83 simple sequence repeats (SSRs) and 50 repeated structures with the highest proportion in the LSC. Comparative analysis of the chloroplast genomes across IRLC revealed three hotspot genes (ycf1, ycf2, clpP) which could be used as DNA barcode regions. Moreover, seven hypervariable regions [trnL(UAA)-trnT(UGU), trnT(GGU)-trnE(UUC), ycf1, ycf2, ycf4, accD and clpP] were identified within Onobrychis, which could be used to distinguish the Onobrychis species. Phylogenetic analyses revealed that O. gaubae is closely related to Hedysarum. The complete O. gaubae genome is a valuable resource for investigating evolution of Onobrychis species and can be used to identify related species. CONCLUSIONS: Our results reveal that the plastomes of the IRLC are dynamic molecules and show multiple gene losses and inversions. The identified hypervariable regions could be used as molecular markers for resolving phylogenetic relationships and species identification and also provide new insights into plastome evolution across IRLC.

Extensive survey of the ycf4 plastid gene throughout the IRLC legumes: Robust evidence of its locus and lineage specific accelerated rate of evolution, pseudogenization and gene loss in the tribe Fabeae.

The genome organization and gene content of plastome (plastid genome) are highly conserved among most flowering plant species. Plastome variation (in size and gene order) is rare in photosynthetic species but size variation, rearrangements and gene/intron losses is attributed to groups of seed plants. Fabaceae (legume family), in particular the subfamily Papilionoideae and the inverted repeat lacking clade (IRLC), a largest legume lineage, display the most dramatic and structural change which providing an excellent model for understanding of mechanisms of genomic evolution. The IRLC comprises 52 genera and ca 4000 species divided into seven tribes. In present study, we have sampled several representatives from each tribe across the IRLC from various herbaria and field. The ycf4 gene, which plays a role in regulating and assembly of photosystem I, is more variable in the tribe Fabeae than in other tribes. In certain species of Lathyrus, Pisum and Vavilovia, all belonging to Fabeae, the gene is either absent or a pseudogene. Our study suggests that ycf4 gene has undergone positive selection. Furthermore, the rapid evolution of the gene is locus and lineage specific and is not a shared character of the IRLC in legumes.

Morphological Innovations and Vast Extensions of Mountain Habitats Triggered Rapid Diversification Within the Species-Rich Irano-Turanian Genus Acantholimon (Plumbaginaceae).

The Irano-Turanian floristic region spans a topographically complex and climatically continental territory, which has served as a source of xerophytic taxa for neighboring regions and is represented by a high percent of endemics. Yet, a comprehensive picture of the abiotic and biotic factors that have driven diversification within this biota remains to be established due to the scarcity of phylogenetic studies. Acantholimon is an important component of the subalpine steppe flora of the Irano-Turanian region, containing c. 200 cushion-forming sub-shrubby pungent-leaved species. Our recent molecular phylogenetic study has led to enlarging the circumscription of this genus to include eight mono- or oligospecific genera lacking the characteristic life-form and leaves. Using the same molecular phylogeny, here we investigate the tempo and mode of diversification as well as the biogeographic patterns in this genus, to test the hypothesis that a combination of key morphological innovations and abiotic factors is behind Acantholimon high species diversity. Molecular dating analysis indicates that Acantholimon s.l. started to diversify between the Late Miocene and the Pliocene and the biogeographic analysis points to an Eastern Iran-Afghanistan origin. Macroevolutionary models support the hypothesis that the high diversity of the genus is explained by accelerated diversification rates in two clades associated with the appearance of morphological key innovations such as a cushion life-form and pungent leaves; this would have favored the colonization of water-stressed, substrate-poor mountainous habitats along the newly uplifted IT mountains during the Mio-Pliocene. Given the apparent similarity of mountain habitats for most species of Acantholimon, we hypothesize that its current high species diversity responds to a scenario of non-adaptive radiation fueled by allopatric speciation rather than evolutionary radiation driven by ecological opportunity. Similar scenarios might underlie the high diversity of other speciose genera in the topographically complex Irano-Turanian landscape, though this remains to be tested with fine-grained distribution and climatic data.

Inhibition of the mevalonate pathway enhances carvacrol biosynthesis and DXR gene expression in shoot cultures of Satureja khuzistanica Jamzad.

Carvacrol is a major component of Satureja khuzistanica Jamzad (≤90%) that has significant antimicrobial and antioxidant properties. Considering the specific capabilities of S. khuzistanica to produce highly pure carvacrol, this plant is an important potential source of carvacrol that could address the abundant consumption and increasing demand for this monoterpene in current world markets. This research was performed to better understand the process of biosynthesis and accumulation of carvacrol in S. khuzistanica. Tests were performed on shoot cultures of S. khuzistanica in Linsmaier-Skoog (LS) medium treated with different concentrations of fosmidomycin (an inhibitor of the non-mevalonate pathway) and mevinolin (an inhibitor of the mevalonate pathway) for 21 days at the following concentrations: 0, 10, 25, 50, 75 and 100 µM. The present study demonstrated that the MEP pathway is the major pathway that provides IPP for the biosynthesis of carvacrol, and the expression and activity levels of the DXR enzyme have a critical effect on carvacrol biosynthesis. Surprisingly, Mevinolin at concentrations of 75 and 100 µM increased the carvacrol content and the DXR activity and gene expression in S. khuzistanica plantlets.

Chemotaxonomic significance of the essential oils of 18 Ferula species (Apiaceae) from Iran.

To evaluate the chemotaxonomic significance of the essential oils of 23 populations of 18 Iranian Ferula species, the chemical composition of the oils was investigated by GC/FID and GC/MS. Altogether, 84 constituents, representing 81.3-99.7% of the total composition of the oils, have been identified. The composition of six species of the genus, i.e., F. oopoda, F. foetida, F. behboudiana, F. diversivittata, F. galbaniflua, and F. hezarlalehzarica, has been reported for the first time. The main constituents identified were α-terpinyl acetate (73.3%), 2,3,4-trimethylthiophene (2; 49.0%), sabinene (75.3%), verbenone (5; 69.4%), ß-pinene (59.0-66.3%), and (Z)-ß-ocimene (41.7%). Cluster analysis (CA) of the percentage content of the essential oil components of the Ferula species resulted in the characterization of four groups, i.e., taxa containing either i) monoterpene hydrocarbons, ii) oxygenated monoterpenes, iii) organosulfur compounds, or iv) monoterpene, sesquiterpene, and aliphatic hydrocarbons as the principal classes of compounds. Based on the results obtained, the chemical independence of F. hirtella from F. szowitsiana and of F. galbaniflua from F. gummosa at the specific level was concluded and their positions as distinct species were confirmed. The chemotaxonomic relationships among the representatives of the genus Ferula have been discussed in detail.