Metabolomic and transcriptomic analyses of yellow-flowered crocuses to infer alternative sources of saffron metabolites.

BACKGROUND: The increasing demand for saffron metabolites in various commercial industries, including medicine, food, cosmetics, and dyeing, is driven by the discovery of their diverse applications. Saffron, derived from Crocus sativus stigmas, is the most expensive spice, and there is a need to explore additional sources to meet global consumption demands. In this study, we focused on yellow-flowering crocuses and examined their tepals to identify saffron-like compounds. RESULTS: Through metabolomic and transcriptomic approaches, our investigation provides valuable insights into the biosynthesis of compounds in yellow-tepal crocuses that are similar to those found in saffron. The results of our study support the potential use of yellow-tepal crocuses as a source of various crocins (crocetin glycosylated derivatives) and flavonoids. CONCLUSIONS: Our findings suggest that yellow-tepal crocuses have the potential to serve as a viable excessive source of some saffron metabolites. The identification of crocins and flavonoids in these crocuses highlights their suitability for meeting the demands of various industries that utilize saffron compounds. Further exploration and utilization of yellow-tepal crocuses could contribute to addressing the growing global demand for saffron-related products.

Plant migration under long-lasting hyperaridity - phylogenomics unravels recent biogeographic history in one of the oldest deserts on Earth.

The post-Miocene climatic histories of arid environments have been identified as key drivers of dispersal and diversification. Here, we investigate how climatic history correlates with the historical biogeography of the Atacama Desert genus Cristaria (Malvaceae). We analyze phylogenetic relationships and historical biogeography by using next-generation sequencing (NGS), molecular clock dating, Dispersal Extinction Cladogenesis and Bayesian sampling approaches. We employ a novel way to identify biogeographically meaningful regions as well as a rarely utilized program permitting the use of dozens of ancestral areas. Partial incongruence between the established taxonomy and our phylogenetic data argue for a complex historical biogeography with repeated introgression and incomplete lineage sorting. Cristaria originated in the central southern part of the Atacama Desert, from there the genus colonized other areas from the late Miocene onwards. The more recently diverged lineages appear to have colonized different habitats in the Atacama Desert during pluvial phases of the Pliocene and early Pleistocene. We show that NGS combined with near-comprehensive sampling can provide an unprecedented degree of phylogenetic resolution and help to correlate the historical biogeography of plant communities with cycles of arid and pluvial phases.

Genome-wide sequence information reveals recurrent hybridization among diploid wheat wild relatives.

Many conflicting hypotheses regarding the relationships among crops and wild species closely related to wheat (the genera Aegilops, Amblyopyrum, and Triticum) have been postulated. The contribution of hybridization to the evolution of these taxa is intensely discussed. To determine possible causes for this, and provide a phylogeny of the diploid taxa based on genome-wide sequence information, independent data were obtained from genotyping-by-sequencing and a target-enrichment experiment that returned 244 low-copy nuclear loci. The data were analyzed using Bayesian, likelihood and coalescent-based methods. D statistics were used to test if incomplete lineage sorting alone or together with hybridization is the source for incongruent gene trees. Here we present the phylogeny of all diploid species of the wheat wild relatives. We hypothesize that most of the wheat-group species were shaped by a primordial homoploid hybrid speciation event involving the ancestral Triticum and Am. muticum lineages to form all other species except Ae. speltoides. This hybridization event was followed by multiple introgressions affecting all taxa except Triticum. Mostly progenitors of the extant species were involved in these processes, while recent interspecific gene flow seems insignificant. The composite nature of many genomes of wheat-group taxa results in complicated patterns of diploid contributions when these lineages are involved in polyploid formation, which is, for example, the case for tetraploid and hexaploid wheats. Our analysis provides phylogenetic relationships and a testable hypothesis for the genome compositions in the basic evolutionary units within the wheat group of Triticeae.

Genomic, karyological and morphological changes of South American garlics (Ipheion) provide insights into mechanisms of speciation in the Pampean region.

Speciation proceeds through mechanisms that promote reproductive isolation and shape the extent of genetic variation in natural populations, and thus its study is essential to understand the evolutionary processes leading to increased biodiversity. Chromosomal rearrangements are known to facilitate reproductive isolation by hybrid sterility and favour speciation events. The genus Ipheion (Amaryllidaceae, Allioideae) is unique as its species exhibit a remarkable karyological variability but lack population-level genetic data. To unveil the diversification processes acting upon the formation of new lineages within Ipheion in the Pampas of South America, we combined morphology and karyology approaches with genotyping-by-sequencing. Our phylogenomic and population genomics results supported the taxonomic division of Ipheion into three morphological and genetically well-differentiated groups. The origin of Ipheion uniflorum was traced back to its current southern distribution area in the southern Pampean region (in Argentina), from where it had expanded to the north reaching Uruguay. Our results further suggested that chromosome rearrangements and ploidy shifts had triggered speciation events, first during the origin of I. uniflorum and later during its subsequent diversification into I. recurvifolium and I. tweedieanum, in both cases reinforced by extrinsic factors and biogeographical settings. The current study illustrates the analytical power of multidisciplinary approaches integrating phylo- and population genomics with classic analyses to reveal evolutionary processes in plants.

Resolving relationships in an exceedingly young Neotropical orchid lineage using Genotyping-by-sequencing data.

Poor morphological and molecular differentiation in recently diversified lineages is a widespread phenomenon in plants. Phylogenetic relationships within such species complexes are often difficult to resolve because of the low variability in traditional molecular loci. Furthermore, biological phenomena responsible for topological incongruence such as Incomplete Lineage Sorting (ILS) and hybridisation complicate the resolution of phylogenetic relationships among closely related taxa. In this study, we employ a Genotyping-by-sequencing (GBS) approach to disentangle evolutionary relationships within a species complex belonging to the Neotropical orchid genus Cycnoches. This complex includes seven taxa distributed through Central America and the Colombian Chocó, and is nested within a clade estimated to have first diversified in the early Quaternary. Previous phylogenies inferred from few loci failed to provide support for internal relationships within the complex. Our Neighbour-net and coalescent-based analyses inferred from ca. 13,000 GBS loci obtained from 31 individuals belonging to six of the seven traditionally accepted Cycnoches taxa provided a robust phylogeny for this group. The genus Cycnoches includes three main clades that are further supported by morphological traits and geographic distributions. Similarly, a topology reconstructed through maximum likelihood (ML) inference of concatenated GBS loci produced results that are comparable with those reconstructed through coalescence and network-based methods. Our comparative phylogenetic informativeness analyses suggest that the low support evident in the ML phylogeny might be attributed to the abundance of uninformative GBS loci, which can account for up to 50% of the total number of loci recovered. The phylogenomic framework provided here, as well as morphological evidence and geographical patterns, suggest that the six entities previously thought to be different species or subspecies might actually represent only three distinct segregates. We further discuss the limited phylogenetic informativeness found in our GBS approach and its utility to disentangle relationships within recent and rapidly evolving species complexes. Our study is the first to demonstrate the utility of GBS data to reconstruct relationships within young (~2 Ma) Neotropical plant clades, opening new avenues for studies of species complexes that populate the species-rich orchid family.

Multiple horizontal transfers of nuclear ribosomal genes between phylogenetically distinct grass lineages.

The movement of nuclear DNA from one vascular plant species to another in the absence of fertilization is thought to be rare. Here, nonnative rRNA gene [ribosomal DNA (rDNA)] copies were identified in a set of 16 diploid barley (Hordeum) species; their origin was traceable via their internal transcribed spacer (ITS) sequence to five distinct Panicoideae genera, a lineage that split from the Pooideae about 60 Mya. Phylogenetic, cytogenetic, and genomic analyses implied that the nonnative sequences were acquired between 1 and 5 Mya after a series of multiple events, with the result that some current Hordeum sp. individuals harbor up to five different panicoid rDNA units in addition to the native Hordeum rDNA copies. There was no evidence that any of the nonnative rDNA units were transcribed; some showed indications of having been silenced via pseudogenization. A single copy of a Panicum sp. rDNA unit present in H. bogdanii had been interrupted by a native transposable element and was surrounded by about 70 kbp of mostly noncoding sequence of panicoid origin. The data suggest that horizontal gene transfer between vascular plants is not a rare event, that it is not necessarily restricted to one or a few genes only, and that it can be selectively neutral.

Elucidation of the origin of 'agriocrithon' based on domestication genes questions the hypothesis that Tibet is one of the centers of barley domestication.

Wild barley forms a two-rowed spike with a brittle rachis whereas domesticated barley has two- or six-rowed spikes with a tough rachis. Like domesticated barley, 'agriocrithon' forms a six-rowed spike; however, the spike is brittle as in wild barley, which makes the origin of agriocrithon obscure. Haplotype analysis of the Six-rowed spike 1 (vrs1) and Non-brittle rachis 1 (btr1) and 2 (btr2) genes was conducted to infer the origin of agriocrithon barley. Some agriocrithon barley accessions (eu-agriocrithon) carried Btr1 and Btr2 haplotypes that are not found in any cultivars, implying that they are directly derived from wild barley through a mutation at the vrs1 locus. Other agriocrithon barley accessions (pseudo-agriocrithon) carried Btr1 or Btr2 from cultivated barley, thus implying that they originated from hybridization between six-rowed landraces carrying btr1Btr2 and Btr1btr2 genotypes followed by recombination to produce Btr1Btr2. All materials we collected from Tibet belong to pseudo-agriocrithon and thus do not support the Tibetan Plateau as being a center of barley domestication. Tracing the evolutionary history of these allelic variants revealed that eu-agriocrithon represents six-rowed barley lineages that were selected by early farmers, once in south-eastern Turkmenistan (vrs1.a1) and again in the eastern part of Uzbekistan (vrs1.a4).

Saffron (Crocus sativus) is an autotriploid that evolved in Attica (Greece) from wild Crocus cartwrightianus.

Crocus sativus, the saffron crocus, is the source of saffron, which is made from the dried stigmas of the plant. It is a male-sterile triploid lineage that ever since its origin has been propagated vegetatively. Its mode of evolution and area of origin are matters of long-lasting debates. Here we analyzed chloroplast genomes and genome-wide DNA polymorphisms obtained through genotyping-by-sequencing (GBS) to infer the parent and area of origin of C. sativus. These data were complemented by genome size measurements and analyses of nuclear single-copy genes. We could place 99.3% of saffron GBS alleles in Crocus cartwrightianus, a species occurring in southeastern mainland Greece and on Aegean islands, identifying it as the sole progenitor of the saffron crocus. Phylogenetic and population assignment analyses together with chloroplast polymorphisms indicated the C. cartwrightianus population in the vicinity of Athens as most similar to C. sativus. We conclude that the crop is an autotriploid that evolved in Attica by combining two different genotypes of C. cartwrightianus. Triploid sterility and vegetative propagation prevented afterwards segregation of the favorable traits of saffron, resulting in worldwide cultivation of a unique clonal lineage.

Phylogeny of the saffron-crocus species group, Crocus series Crocus (Iridaceae).

Phylogenetic relationships among the taxa of Crocus series Crocus are still unclear, preventing the understanding of species diversity and the evolution of the important spice saffron (Crocus sativus). Therefore, we analyzed sequences of two chloroplast (trnL-trnF, matK-trnK) and three nuclear (TOPO6, ribosomal DNA ETS and ITS) marker regions to infer phylogenetic relationships among all species belonging to series Crocus. Our phylogenetic analyses resolved the relationships among all taxa of the series. Crocus hadriaticus and the former C. pallasii subspecies appeared polyphyletic. The latter deserve elevating the subspecies to species rank, while for C. hadriaticus a detailed study of species boundaries is necessary. Multi-locus and also genome-wide single nucleotide polymorphism data obtained through genotyping-by-sequencing placed C. sativus within C. cartwrightianus with no indication that other Crocus species contributed to the evolution of the triploid. Our analyses thus made an autotriploid origin of C. sativus from C. cartwrightianus very likely.

Dated tribe-wide whole chloroplast genome phylogeny indicates recurrent hybridizations within Triticeae.

BACKGROUND: Triticeae, the tribe of wheat grasses, harbours the cereals barley, rye and wheat and their wild relatives. Although economically important, relationships within the tribe are still not understood. We analysed the phylogeny of chloroplast lineages among nearly all monogenomic Triticeae taxa and polyploid wheat species aiming at a deeper understanding of the tribe's evolution. We used on- and off-target reads of a target-enrichment experiment followed by Illumina sequencing. RESULTS: The read data was used to assemble the plastid locus ndhF for 194 individuals and the whole chloroplast genome for 183 individuals, representing 53 Triticeae species and 15 genera. We conducted Bayesian and multispecies coalescent analyses to infer relationships and estimate divergence times of the taxa. We present the most comprehensive dated Triticeae chloroplast phylogeny and review previous hypotheses in the framework of our results. Monophyly of Triticeae chloroplasts could not be confirmed, as either Bromus or Psathyrostachys captured a chloroplast from a lineage closely related to a Bromus-Triticeae ancestor. The most recent common ancestor of Triticeae occurred approximately between ten and 19 million years ago. CONCLUSIONS: The comparison of the chloroplast phylogeny with available nuclear data in several cases revealed incongruences indicating past hybridizations. Recent events of chloroplast capture were detected as individuals grouped apart from con-specific accessions in otherwise monopyhletic groups.

Repair of Site-Specific DNA Double-Strand Breaks in Barley Occurs via Diverse Pathways Primarily Involving the Sister Chromatid.

DNA double-strand break (DSB) repair mechanisms differ in their requirements for a homologous repair template and in the accuracy of the result. We aimed to quantify the outcome of repair of a single targeted DSB in somatic cells of young barley (Hordeum vulgare) plants. Amplicon sequencing of three reporter constructs revealed 47 to 58% of reads as repaired via nonhomologous end-joining (NHEJ) with deletions and/or small (1 to 3 bp) insertions. Alternative NHEJ revealed 2 to 5 bp microhomology (15.7% of cases) or new replication-mediated short duplications at sealed breaks. Although deletions outweigh insertions in barley, this bias was less pronounced and deleted sequences were shorter than in Arabidopsis thaliana. Between 17 and 33% of reads likely represent restoration of the original sequence. Depending on the construct, 20 to 33% of reads arose via gene conversion (homologous recombination). Remarkably, <1 to >8% of reads apparently display synthesis-dependent strand annealing linked with NHEJ, inserting 4 to 61 bp, mostly originating from the surrounding of breakpoints. Positional coincidence of >81% of sister chromatid exchanges with target loci is unprecedented for higher eukaryotes and indicates that most repair events for staggered DSBs, at least in barley, involve the sister chromatid and occur during S or G2 phase of the cell cycle.

Species-Level Phylogeny and Polyploid Relationships in Hordeum (Poaceae) Inferred by Next-Generation Sequencing and In Silico Cloning of Multiple Nuclear Loci.

Polyploidization is an important speciation mechanism in the barley genus Hordeum. To analyze evolutionary changes after allopolyploidization, knowledge of parental relationships is essential. One chloroplast and 12 nuclear single-copy loci were amplified by polymerase chain reaction (PCR) in all Hordeum plus six out-group species. Amplicons from each of 96 individuals were pooled, sheared, labeled with individual-specific barcodes and sequenced in a single run on a 454 platform. Reference sequences were obtained by cloning and Sanger sequencing of all loci for nine supplementary individuals. The 454 reads were assembled into contigs representing the 13 loci and, for polyploids, also homoeologues. Phylogenetic analyses were conducted for all loci separately and for a concatenated data matrix of all loci. For diploid taxa, a Bayesian concordance analysis and a coalescent-based dated species tree was inferred from all gene trees. Chloroplast matK was used to determine the maternal parent in allopolyploid taxa. The relative performance of different multilocus analyses in the presence of incomplete lineage sorting and hybridization was also assessed. The resulting multilocus phylogeny reveals for the first time species phylogeny and progenitor-derivative relationships of all di- and polyploid Hordeum taxa within a single analysis. Our study proves that it is possible to obtain a multilocus species-level phylogeny for di- and polyploid taxa by combining PCR with next-generation sequencing, without cloning and without creating a heavy load of sequence data.

Oligocene niche shift, Miocene diversification - cold tolerance and accelerated speciation rates in the St. John's Worts (Hypericum, Hypericaceae).

BACKGROUND: Our aim is to understand the evolution of species-rich plant groups that shifted from tropical into cold/temperate biomes. It is well known that climate affects evolutionary processes, such as how fast species diversify, species range shifts, and species distributions. Many plant lineages may have gone extinct in the Northern Hemisphere due to Late Eocene climate cooling, while some tropical lineages may have adapted to temperate conditions and radiated; the hyper-diverse and geographically widespread genus Hypericum is one of these. RESULTS: To investigate the effect of macroecological niche shifts on evolutionary success we combine historical biogeography with analyses of diversification dynamics and climatic niche shifts in a phylogenetic framework. Hypericum evolved cold tolerance c. 30 million years ago, and successfully colonized all ice-free continents, where today ~500 species exist. The other members of Hypericaceae stayed in their tropical habitats and evolved into ~120 species. We identified a 15-20 million year lag between the initial change in temperature preference in Hypericum and subsequent diversification rate shifts in the Miocene. CONCLUSIONS: Contrary to the dramatic niche shift early in the evolution of Hypericum most extant species occur in temperate climates including high elevations in the tropics. These cold/temperate niches are a distinctive characteristic of Hypericum. We conclude that the initial release from an evolutionary constraint (from tropical to temperate climates) is an important novelty in Hypericum. However, the initial shift in the adaptive landscape into colder climates appears to be a precondition, and may not be directly related to increased diversification rates. Instead, subsequent events of mountain formation and further climate cooling may better explain distribution patterns and species-richness in Hypericum. These findings exemplify important macroevolutionary patterns of plant diversification during large-scale global climate change.

Selfish supernumerary chromosome reveals its origin as a mosaic of host genome and organellar sequences.

Supernumerary B chromosomes are optional additions to the basic set of A chromosomes, and occur in all eukaryotic groups. They differ from the basic complement in morphology, pairing behavior, and inheritance and are not required for normal growth and development. The current view is that B chromosomes are parasitic elements comparable to selfish DNA, like transposons. In contrast to transposons, they are autonomously inherited independent of the host genome and have their own mechanisms of mitotic or meiotic drive. Although B chromosomes were first described a century ago, little is known about their origin and molecular makeup. The widely accepted view is that they are derived from fragments of A chromosomes and/or generated in response to interspecific hybridization. Through next-generation sequencing of sorted A and B chromosomes, we show that B chromosomes of rye are rich in gene-derived sequences, allowing us to trace their origin to fragments of A chromosomes, with the largest parts corresponding to rye chromosomes 3R and 7R. Compared with A chromosomes, B chromosomes were also found to accumulate large amounts of specific repeats and insertions of organellar DNA. The origin of rye B chromosomes occurred an estimated ∼1.1-1.3 Mya, overlapping in time with the onset of the genus Secale (1.7 Mya). We propose a comprehensive model of B chromosome evolution, including its origin by recombination of several A chromosomes followed by capturing of additional A-derived and organellar sequences and amplification of B-specific repeats.

Barley whole exome capture: a tool for genomic research in the genus Hordeum and beyond.

Advanced resources for genome-assisted research in barley (Hordeum vulgare) including a whole-genome shotgun assembly and an integrated physical map have recently become available. These have made possible studies that aim to assess genetic diversity or to isolate single genes by whole-genome resequencing and in silico variant detection. However such an approach remains expensive given the 5 Gb size of the barley genome. Targeted sequencing of the mRNA-coding exome reduces barley genomic complexity more than 50-fold, thus dramatically reducing this heavy sequencing and analysis load. We have developed and employed an in-solution hybridization-based sequence capture platform to selectively enrich for a 61.6 megabase coding sequence target that includes predicted genes from the genome assembly of the cultivar Morex as well as publicly available full-length cDNAs and de novo assembled RNA-Seq consensus sequence contigs. The platform provides a highly specific capture with substantial and reproducible enrichment of targeted exons, both for cultivated barley and related species. We show that this exome capture platform provides a clear path towards a broader and deeper understanding of the natural variation residing in the mRNA-coding part of the barley genome and will thus constitute a valuable resource for applications such as mapping-by-sequencing and genetic diversity analyzes.

Phylogeny of Crocus (Iridaceae) based on one chloroplast and two nuclear loci: ancient hybridization and chromosome number evolution.

Crocus consists of about 100 species distributed from western Europe and northern Africa to western China, with the center of diversity on the Balkan Peninsula and in Asia Minor. Our study focuses on clarifying phylogenetic relationships and chromosome number evolution within the genus using sequences of the chloroplast trnL-F region, the nuclear ribosomal DNA internal transcribed spacer (ITS) region, and a part of the nuclear single-copy gene pCOSAt103. In a combined dataset of ITS and trnL-F sequences, 115 individuals representing 110 taxa from both subgenera and all sections and series of Crocus were analyzed with Bayesian phylogenetic inference. For pCOSAt103 79 individuals representing 74 Crocus taxa were included, and for the majority of them PCR amplicons were cloned and up to eight clones per individual were sequenced to detect allopolyploidization events. Romulea species were included as outgroup in both analyses. Characteristics of seed surface structures were evaluated by scanning electron microscopy. Phylogenetic analysis of ITS/trnL-F data resulted in a monophyletic genus Crocus, probably monophyletic sections Crocus and Nudiscapus, and inferred monophyly for eight of the 15 series of the genus. The C. biflorus aggregate, thought to be consisting of closely related subspecies, was found to be polyphyletic, the taxa occurring within three major clades in the phylogenetic tree. Cloning of pCOSAt103 resulted in the detection of homoeologous copies in about one third of the taxa of section Nudiscapus, indicating an allotetraploid origin of this section. Reconstruction of chromosome number evolution along the phylogenetic tree using a probabilistic and a parsimony approach arrived at partly contradictory results. Both analyses agreed however on the occurrence of multiple polyploidization and dysploidy events. B chromosomes evolved at least five times independently within the genus, preferentially in clades characterized by karyotype changes.

Molecular phylogenetics and morphological evolution of St. John's wort (Hypericum; Hypericaceae).

Phylogenetic hypotheses for the large cosmopolitan genus Hypericum (St. John's wort) have previously been based on morphology, and molecular studies have thus far included only a few species. In this study, we used 360 sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) for 206 species representing Hypericum (incl. Triadenum and Thornea) and three other genera of Hypericaceae to generate an explicit phylogenetic hypothesis for the genus using parsimony and model-based methods. The results indicate that the small genus Triadenum is nested in a clade within Hypericum containing most of the New World species. Sister to Hypericum is Thornea from Central America. Within Hypericum, three large clades and two smaller grades were found; these are based on their general morphology, especially characters used previously in taxonomy of the genus. Relative to the most recent classification, around 60% of the sections of Hypericum were monophyletic. We used a Bayesian approach to reconstruct ancestral states of selected morphological characters, which resulted in recognition of characters that support major clades within the genus and a revised interpretation of morphological evolution in Hypericum. The shrubby habit represents the plesiomorphic state from which herbs evolved several times. Arborescent species have radiated convergently in high-elevation habitats in tropical Africa and South America.

B chromosomes of rye are highly conserved and accompanied the development of early agriculture.

BACKGROUND AND AIMS: Supernumerary B chromosomes (Bs) represent a specific type of selfish genetic element. As Bs are dispensable for normal growth, it is expected to observe B polymorphisms among populations. To address whether Bs maintained in geographically distinct populations of cultivated and weedy rye are polymorphic, the distribution patterns and the transcriptional activity of different B-located repeats were analysed. METHODS: Bs of cultivated and weedy rye from seven origins were analysed by fluorescence in situ hybridization (FISH) with probes specific for the pericentromeric and interstitial regions as well as the B-specific non-disjunction control region. The DNA replication, chromatin composition and transcription behaviour of the non-disjunction regions were determined. To address whether the B-marker repeats E3900 and D1100 have diverged genotypes of different origin at the sequence level, the genomic sequences of both repeats were compared between cultivated rye and weedy rye from five different origins. KEY RESULTS: B chromosomes in cultivated and weedy rye have maintained a similar molecular structure at the level of subspecies. The high degree of conservation of the non-disjunction control region regarding its transcription activity, histone composition and replication underlines the functional importance of this chromosome region for the maintenance of Bs. The conserved chromosome structure suggests a monophyletic origin of the rye B. As Bs were found in different countries, it is likely that Bs were frequently present in the seed material used in early agriculture. CONCLUSIONS: The surprisingly conserved chromosome structure suggests that although the rye Bs experienced rapid evolution including multiple rearrangements at the early evolutionary stages, this process has slowed significantly and may have even ceased during its recent evolution.

Dated Phylogeny of Astragalus Section Stereothrix (Fabaceae) and Allied Taxa in the Hypoglottis Clade.

The Astragalus subgenus Hypoglottis Bunge, which consists of several sections, is one of the taxonomically most complicated groups in the genus. The Astragalus section Stereothrix Bunge belongs to this subgenus and is a significant element of the Irano-Turanian floristic region. A molecular phylogenetic analysis of this section and its closely related taxa using nuclear ribosomal DNA internal transcribed spacers (ITS) and external transcribed spacer (ETS) regions as well as plastid matK sequences were conducted. Parsimony analyses and Bayesian phylogenetic inference revealed that the section is not monophyletic in its current form, as some taxa belonging to closely related sections such as Hypoglottidei DC. and the Malacothrix Bunge group within the sect. Stereothrix render it paraphyletic. Moreover, species groups belonging to sect. Stereothrix are placed in different clades within the phylogenetic tree of subgenus Hypoglottis, which indicates polyphyly, i.e., multiple independent origins of taxa placed in the sect. Stereothrix. Molecular dating of the group estimated an age of 3.62 (1.73-5.62) My for this assemblage with the major diversification events happening during the last 2 My. Many species groups separated only within the last 0.5 to 1 My. Based on morphological and molecular data, we discuss the phylogenetic relationships of the groups and synonymy of species. In addition, the included taxa of sect. Hypoglottidei are not monophyletic and include species belonging to sects. Hololeuce, Koelziana, Malacothrix, Onobrychoideae, and Ornithodpodium group within the sect. Stereothrix taxa. We conclude that only an analysis including all groups and nearly all species of the sections within the Hypoglottis clade can finally result in an new evolutionary-based system for these taxa.

Disentangling Crocus Series Verni and Its Polyploids.

Spring crocuses, the eleven species within Crocus series Verni (Iridaceae), consist of di- and tetraploid cytotypes. Among them is a group of polyploids from southeastern Europe with yet-unclear taxonomic affiliation. Crocuses are generally characterized by complex dysploid chromosome number changes, preventing a clear correlation between these numbers and ploidy levels. To reconstruct the evolutionary history of series Verni and particularly its polyploid lineages associated with C. heuffelianus, we used an approach combining phylogenetic analyses of two chloroplast regions, 14 nuclear single-copy genes plus rDNA spacers, genome-wide genotyping-by-sequencing (GBS) data, and morphometry with ploidy estimations through genome size measurements, analysis of genomic heterozygosity frequencies and co-ancestry, and chromosome number counts. Chromosome numbers varied widely in diploids with 2n = 8, 10, 12, 14, 16, and 28 and tetraploid species or cytotypes with 2n = 16, 18, 20, and 22 chromosomes. Crocus longiflorus, the diploid with the highest chromosome number, possesses the smallest genome (2C = 3.21 pg), while the largest diploid genomes are in a range of 2C = 7-8 pg. Tetraploid genomes have 2C values between 10.88 pg and 12.84 pg. Heterozygosity distribution correlates strongly with genome size classes and allows discernment of di- and tetraploid cytotypes. Our phylogenetic analyses showed that polyploids in the C. heuffelianus group are allotetraploids derived from multiple and partly reciprocal crosses involving different genotypes of diploid C. heuffelianus (2n = 10) and C. vernus (2n = 8). Dysploid karyotype changes after polyploidization resulted in the tetraploid cytotypes with 20 and 22 chromosomes. The multi-data approach we used here for series Verni, combining evidence from nuclear and chloroplast phylogenies, genome sizes, chromosome numbers, and genomic heterozygosity for ploidy estimations, provides a way to disentangle the evolution of plant taxa with complex karyotype changes that can be used for the analysis of other groups within Crocus and beyond. Comparing these results with morphometric analysis results in characters that can discern the different taxa currently subsumed under C. heuffelianus.