Genus Crocus Research Articles

Seed structure of some taxa of the genus Crocus L. (Iridaceae) series Crocus

In this study, the macro- and micromorphological seed characteristics of the genus Crocus L. series Crocus taxa, including C. pallasii , C. turcicus , C. dispathaceus , C. asumaniae , C. mathewii , C. thomasii , C. oreocreticus , and C. cartwrightianus , were observed. Morphological characteristics, such as the size, shape, color, and seed surface structures, were analyzed using stereo and scanning electron microscopes. Testa layer structures including the outer and inner testae, phytomelan layer structure and thickness, and seed endosperm cell features were studied. Differences in the seed, raphe size, seed surface patterns, inner testa features, and phytomelan layer thickness were observed at an interspecific level. The results revealed differences in the seed morphology and anatomy with a few exceptions.

Centaurea francoi, a replacement name for Centaurea crocata (Asteraceae), an endemic species from continental Portugal

Centaurea crocata Franco (1984: 474, 572) is the name currently used for a species of Asteraceae that is endemic to a small area in the southwest of Portugal, extending from Monchique (Algarve) to Cercal (Baixo Alentejo). The plant was first collected by Friedrich Welwitsch in 1847, in Monchique, during his residence (1847–1848) in the province of Algarve (Trimen 1873: 3). The following year he collected it again but further north, in Baixo Alentejo. Welwitsch regarded it as a new species and informally named it ‘Centaurea crocea’, as is apparent from the name written on his specimens of this species that are held in LISU (Garcia Jacas & Susanna 1991). However, he never published this name. Later authors working on the flora of Portugal, such as Sampaio (1909: 60, 1947: 597) and Coutinho (1913: 657, 1939: 776) misidentified Welwitsch’s and other collectors’ material as C. prolongoi Boissier ex Candolle (1838: 303, originally published as ‘prolongi’, corrected to ‘prolongoi’ as it commemorates the Spanish botanist Pablo Prolongo y Garcia). It was only much later that Franco (1984), in the second volume of his Flora of Portugal, concurred with Welwitsch’s view that the material belonged to a separate species and described it as C. crocata, using an epithet similar to that of Welwitsch’s unpublished name. Both epithets ‘crocata’ and ‘crocea’ originate from Latin and mean saffron-coloured. Saffron is a product of a species of the genus Crocus Linnaeus (1753: 36), a name with the same origin.

A new Serbian endemic species of the genus Crocus (Iridaceae)

Recent research within the genus Crocus (Iridaceae) let us doubt that Crocus adamii Gay from Serbia represents the same taxon as C.adamii s. str. of the locus classicus in the Caucasian Mountains. The latter belongs to a group of crocuses, which is distributed from the Anatolian Diagonal, a mountain belt in inner Anatolia, to Iran and the Caucasian Mountains. To infer ( i ) if the Serbian C. adamii represents a new species and ( ii ) its taxonomical and phylogenetic affiliation within the genus we combined morphological and molecular investigations. The results show the presence of two morphologically and molecularly differentiated lineages, which both share a close relationship e.g. to C. alexandrii , C. chrysanthus , and C. weldenii but not to C. adamii s.str., which indicates a new species. As a result we here describe C. randjeloviciorum to honor the Serbian botanists Novica and Vladimir Ranđelovic.

Novica Ranđelović: Tireless botanist explores on the verge of his ninth decade

Exactly ten years from 9 th Symposium on the Flora of Southeastern Serbia and Neighboring Regions which was organized in honor of 70 th birthday of Novica Randjelovic. He was founder informal South Serbian Botanical School, Symposium on the Flora of Southeastern Serbia and Neighboring Regions, founder and first professor on Department for biology and ecology from University of Nis. Even though he was in eight decades professor Randjelovic was tirelessly continued with research. He published many scientific papers. Especially are important those with description of new species from his favorite genus Crocus - C. jablanicensis (Ranđelovic et al., 2011) i C. danubiensis (Harpke et al., 2014). Two species was named in his honor – Crocus novicii (Miljkovic et al., 2016) and C. randjeloviciorum (Harpke et al., 2017). From 2007. to 2017. he published 13 papers (6 in international journals). Besides that, he is almost regular on botanical scientific symposiums and congresses around Balkan peninsula.

A new species of Crocus (Iridaceae) from southern Albania (SW Balkan Peninsula)

A new Crocus species (Iridaceae) for southern Albania, Crocus novicii sp. nov. (Iridaceae), is described and illustrated. We here provide diagnostic morphological characteristics, results of molecular analyses, detailed descriptions and illustrations of this new species and compare it with its relatives C. jablanicensis, C. cvijicii and C. veluchensis. Our molecular analysis is based on two chloroplast (matK–trnK, rps16–trnQ) and three nuclear markers (nrETS, nrITS, TOPO6B exon3–exon6) and includes representatives of all related species (C. cvijicii, C. dalmaticus, C. jablanicensis, C. rujanensis, C. sieberi subsp. atticus, and C. veluchensis). Morphologically, C. novicii can be distinguished from its relatives by its white flower with lilac coloring at the base of the perigone and its higher number of leaves (> 3). Although it is morphologically close to C. jablanicensis, molecular analysis has revealed a close affiliation to C. veluchensis. Crocus novicii is diploid with 2n = 20 chromosomes. The new species is named after the Serbian botanist Novica Ranđelović to honor his important work on the genus Crocus on the Balkan Peninsula.

Crocus heuffelianus (Iridaceae), a new record for the Italian flora

The genus Crocus Linnaeus (1753: 36) consists currently of about 200 recognized species occurring from western Europe and northwestern Africa to western China, with the center of species diversity in the Balkan Peninsula and in Turkey (Mathew 1982, Harpke et al. 2016). In the most recent comprehensive classification (Mathew 1982), which relied only on morphology, the genus is divided into two subgenera, the monotypic subgenus Crociris Mathew (1982: 116) and subgenus Crocus. The latter is divided into two sections, i.e. section Crocus and section Nudiscapus Mathew (1982: 61), and includes 15 series (section Crocus: six series; section Nudiscapus: nine series).

Neotypification of the name Crocus biflorus Mill. (Iridaceae) and its consequences in the taxonomy of the genus

Recent phylogenetic investigations on the genus Crocus proved several infra-generic units predominantly within section Nudiscapus to be para- or polyphyletic even at infra-specific level. In particular, the 23 “subspecies” of C. biflorus Mill. turned out to be a polyphyletic assemblage grouping in very different clades and sub-groups. In addition, they were established under the binomial Crocus biflorus, not yet typified. As first step, we neotypified this name. Then, through a phylogeny based on the ETS region of the nuclear ribosomal (nr) DNA as an additional nuclear marker to the already available nrITS region, we further demonstrated that all the “subspecies” represent independent evolutionary lineages. Therefore, all these taxa are treated here at species level. To achieve this, 10 new combinations are proposed.

A Matter of Contrast: Yellow Flower Colour Constrains Style Length in Crocus species.

Most flowers display distinct colour patterns comprising two different areas. The peripheral large-area component of floral colour patterns attracts flower visitors from some distance and the central small-area component guides flower visitors towards landing sites. Whereas the peripheral colour is largely variable among species, the central colour, produced mostly by anthers and pollen or pollen mimicking floral guides, is predominantly yellow and UV-absorbing. This holds also for yellow flowers that regularly display a UV bull’s eye pattern. Here we show that yellow-flowering Crocus species are a noticeable exception, since yellow-flowering Crocus species–being entirely UV-absorbing–exhibit low colour contrast between yellow reproductive organs and yellow tepals. The elongated yellow or orange-yellow style of Crocus flowers is a stamen-mimicking structure promoting cross-pollination by facilitating flower visitors’ contact with the apical stigma before the flower visitors are touching the anthers. Since Crocus species possess either yellow, violet or white tepals, the colour contrast between the stamen-mimicking style and the tepals varies among species. In this study comprising 106 Crocus species, it was tested whether the style length of Crocus flowers is dependent on the corolla colour. The results show that members of the genus Crocus with yellow tepals have evolved independently up to twelve times in the genus Crocus and that yellow-flowering Crocus species possess shorter styles as compared to violet- and white-flowering ones. The manipulation of flower visitors by anther-mimicking elongated styles in Crocus flowers is discussed.

Molecular genetic evidence for allotetraploid hybrid speciation in the genus Crocus L. (Iridaceae)

Although dysploidy and polyploidisation events are known to be important drivers in the evolutionary history of the genus Crocus, only a few examples of natural hybrid origins have so far been documented. Here, we describe the phylogenetic affinities of five taxa in the Crocus vernus species complex from the Carpathian Basin in Central Europe. Genetic variability was evaluated using chloroplast DNA sequences of the accD-psaI intergenic spacer and amplified fragment length polymorphism (AFLP) in combination with karyological observations. Genetic diversity and differentiation of the seven investigated Crocus species were also evaluated using AFLP data. We created a phylogenetic hypothesis using both sequences and AFLP fingerprinting data under maximum parsimony (MP). AFLPs were also analysed by means of multivariate statistics using principal coordinate (PCoA) analysis and Bayesian clustering (BC) to test for hybridity. Both the AFLP and plastid phylogenetic trees separated the taxa into two groups: (1) a ‘Balkan’ clade, and; (2) an ‘Adriatic’ clade. The Balkan clade contained Crocus heuffelianus samples and one Crocus vittatus from Croatia as well as Crocus tommasinianus samples from Hungary; the Adriatic clade included Crocus vittatus and Crocus vernus samples from Hungary and Croatia as well as Crocus neapolitanus from Italy. A hard incongruence was found in the placement of the Slovakian Crocus scepusiensis which clustered inside the Balkan clade on our plastid tree, while it fell in the Adriatic clade on the AFLP tree. The same populations occupied intermediate position on the PCoA plot. The BC analysis assigned all Crocus scepusiensis specimens as F1 hybrids, while Crocus vernus and Crocus heuffelianus were assigned as parental species. Together with our cytological investigation that determined a 2n=18 chromosome number of Crocus scepusiensis, we conclude an allopolyploid hybrid origin for this Northern Carpathian taxon as the result of a cross between members of the 2n=10 Balkan clade (e.g., Crocus heuffelianus from Transylvania) and the 2n=8 Adriatic clade (Crocus vernus). A similar origin is postulated for the North Balkan Crocus vittatus (2n=18), which was clustered as an F1 hybrid in the BC analysis; thus, parallel evolution may have taken place in the northern and southern part of the Carpathian Basin beginning from the same parental species but leading to different allopolyploid derivatives.

Exploiting DNA mini-barcodes as molecular markers to authenticate saffron (Crocus sativus L.)

Saffron (Crocus sativus) is the most expensive spice in the world. As a consequence, saffron has frequently been associated with an unrivalled degree of adulteration. The aim of this work was searching for DNA-barcode candidates as molecular markers to differentiate C. sativus from allied species and assessing the authenticity of saffron. New primers were specifically designed targeting three DNA barcode loci, namely ITS1, ITS2 and matK. A real-time PCR assay combined with high resolution melting (HRM) analysis was carried out in order to discriminate different Crocus spp., being further confirmed by sequencing. Data from HRM analysis showed that ITS1 and matK loci were Crocus genus specific, while ITS2 locus was species-specific for C. sativus and Crocus cartwrightianus. This study revealed that genomic tools, and specifically DNA mini-barcodes combined with HRM analysis, can provide simple, fast and highly reliable methods for saffron authentication.

Somatic Embryogenesis in Crocus sativus L.

Saffron (Crocus sativus L.) is one of the most important species in Crocus genus because of its effective usage. It is not only a very expensive spice, but it has also a big ornamental plant potential. Crocus species are propagated by corm and seed, and male sterility is the most important problem of this species. Hence, somatic embryogenesis can be regarded as a strategic tool for the multiplication of saffron plants. In this chapter, the production of saffron corms via somatic embryogenesis is described.

Saffron: Its Phytochemistry, Developmental Processes, and Biotechnological Prospects.

The present state of knowledge concerning developmental processes and the secondary metabolism of saffron, Crocus sativus L. (Iridaceae), along with the genes involved in these processes so far known, is reviewed. Flowers and corms constitute the most valuable parts of saffron. Corm and flower development are two key aspects to be studied in saffron to increase the yield and quality of the spice, to raise its reproductive rate, and to implement new production systems. Important knowledge about the physiology of flowering and vegetative growth has been acquired in recent years, but there is still only limited information on molecular mechanisms controlling these processes. Although some genes involved in flower formation and meristem transition in other species have been isolated in saffron, the role of these genes in this species awaits further progress. Also, genes related with the synthesis pathway of abscisic acid and strigolactones, growth regulators related with bud endodormancy and apical dominance (paradormancy), have been isolated. However, the in-depth understanding of these processes as well as of corm development is far from being achieved. By contrast, saffron phytochemicals have been widely studied. The different flower tissues and the corm have been proved to be an important source of phytochemicals with pharmacological properties. The biotechnological prospects for saffron are here reviewed on the basis of the discovery of the enzymes involved in key aspects of saffron secondary metabolism, and we also analyze the possibility of transferring current knowledge about flowering and vegetative propagation in model species to the Crocus genus.

Neuropharmacological Aspects of Crocus sativus L.: A Review of Preclinical Studies and Ongoing Clinical Research.

Crocus sativus L. (Iridaceae) is an important member of the genus Crocus having high medicinal value. Its dried stigmas, known as "saffron" are being widely used form past many centuries as a food additive, coloring agent, flavoring agent and a potential source of traditional medicine. The stigmas along with other botanical parts of Crocus sativus are being extensively used in ethnomedical treatment of varied central nervous system diseases. In line with its ethnomedical importance, several preclinical studies have been carried out to validate its traditional uses, identify active principle(s), understand pharmacological basis of therapeutic action and explore novel medicinal uses. The bioactive components of Crocus sativus have been found to modulate several synaptic processes via direct/indirect interplay with neurotransmitter receptor functions, interaction with neuronal death/survival pathways and alteration in neuronal proteins expression. Many clinical studies proving beneficial effect of Crocus sativus in depressive disorders, Alzheimer's disease and some other neurological abnormalities have also been carried out. Based on the vast literature reports available, an attempt has been made to comprehend the fragmented information on neuropharmacological aspects, chemistry and safety of Crocus sativus. Although the plant has been well explored, but still a large scope of future preclinical and clinical research exist to explore its potential in neurological diseases, that has been discussed in depth in the present review.

Diversity and relationships of Crocus sativus and its relatives analysed by inter-retroelement amplified polymorphism (IRAP).

Saffron (Crocus sativus) is a sterile triploid (2n = 3x = 24) cultivated species, of unknown origin from other diploid and polyploid species in the genus Crocus (Iridaceae). Species in the genus have high morphological diversity, with no clear phylogenetic patterns below the level of section Crocus series Crocus. Using DNA markers, this study aimed to examine the diversity and relationships within and between species of Crocus series Crocus. Eleven inter-retroelement amplified polymorphism (IRAP) primers were used in 63 different combinations with 35 single-plant accessions of C. sativus and related Crocus species in order to determine genetic variability and to conduct phylogenetic analysis. A total of 4521 distinct polymorphic bands from 100 bp to approx. 4 kb were amplified; no fragment specific to all accessions of a single species was amplified. The polymorphic information content (PIC) values varied from approx. 0·37 to approx. 0·05 (mean 0·17 ± 0·1) and the major allele frequency had a mean of 0·87. High levels of polymorphism were identified between accessions of the six species of Crocus series Crocus related to C. sativus, with further variation between the species. In contrast, no polymorphisms were seen among 17 C. sativus accessions obtained in the region from Kashmir through Iran to Spain. In contrast to the intraspecific variability seen in other Crocus species, C. sativus has minimal genetic variation, and it is concluded that the triploid hybrid species has most probably arisen only once. The data show that saffron is an allotriploid species, with the IRAP analysis indicating that the most likely ancestors are C. cartwrightianus and C. pallasii subsp. pallasii (or close relatives). The results may facilitate resynthesizing saffron with improved characteristics, and show the need for conservation and collection of wild Crocus.

Saffron (Crocus sativus L.), a monomorphic or polymorphic species?

Saffron (Crocus sativus L.) which contains exceptional anti-cancer properties is presently the world's most expensive spice. Iran is known as the original habitat of Crocus L. and a significant source of high-quality cultivated saffron production and export. Considering the importance of this species, we used 27 microsatellite markers to assess molecular variability and discriminating capacity of markers regarding their effectiveness in establishing genetic relationships in Iranian Crocus ecotypes. Thirty eight Iranian cultivated saffron ecotypes and 29 wild allies were evaluated in this research. The results from molecular analyses, including a molecular phylogenetic network and RB analysis, revealed two major groups and five subgroups, regardless of their geographical origins. Also, the results showed a clear distinction between C. sativus and other species of Crocus genus, taking into account their close relationship with C. speciosus and C. hausknechtii, which are assumed to be the two closest relatives of Iranian cultivated saffron among species studied. In this paper, we observed for the first time extensive genetic diversity among Iranian C. sativus despite their asexual reproduction. Considering suitable climatic conditions in Iran for cultivating saffron and the country’s leading high-quality production of Crocus sativus worldwide, studies on great genetic variability among Iranian C. sativus ecotypes as well as wild relatives native to Iran will further highlight the value of this crop. In addition, our results provide valuable information for genetic improvement, reduction of strong genetic erosion, and conservation of costly heritable resources of C. sativus in future breeding programs.

English

Saffron (Crocus sativus L.) is a sterile triploid plant belonging to the Iridaceae (Liliales, Monocots). Saffron is a spice derived from the flower and has for decades been the world's most expensive spice. Saffron is propagated by corms as the flowers are sterile and fail to produce viable seeds. A corm survives for only one season, producing up to ten "cormlets" that eventually give rise to new plants. Therefore, reproduction is human dependent; the corms must be manually dug up, broken apart and replanted. The natural propagation rate of saffron is relatively low. Biotechnological approaches have increasingly become a valuable tool assisting breeders to release new species and cultivars into the market more rapidly. Biotechnological approaches offer the capability to produce large quantities of propagating material in short time as well as the production of commercially important chemical constituents like, crocin, picrocrocin, crocetin and safranal under in vitro conditions. However, the protocols available so far need further refinement for their commercial utilization. Here we review the progress made in genus Crocus, and highlight the potential for future expansion in this field through biotechnological interventions. Key words: Crocus sativus L, biotechnological approaches, sterile, corms.

The genus Crocus, series Crocus (Iridaceae) in Turkey and 2 East Aegean islands: a genetic approach

In this study, a total of 26 Crocus specimens from different locations across Turkey and 2 East Aegean islands (Chios and Samos) were analyzed using 12 amplified fragment length polymorphism (AFLP) primer combinations to obtain information on genetic diversity, population structure, and genetic relationships. A total of 369 polymorphic AFLP bands were generated and scored as binary data. Genetic similarities were determined. Cluster analysis revealed 4 major groups among the 26 genotypes examined in this study. The nuclear DNA contents (2C) of the 26 Crocus specimens were found to range from 5.08 pg in C. asumaniae to 9.75 pg in C. sativus. Polymorphic information content (PIC) values were used to examine the capacity of the various primer pairs to amplify polymorphisms in the Crocus specimens. The PIC values ranged from 0.218 (M-CAA/E-AGC) to 0.512 (M-CAT/E-AAG) and showed an average of 0.34. In sum, we herein used AFLP analysis to identify a high level of polymorphism among Crocus specimens collected from various locations in Turkey and Greece, and our structural analysis yielded 2 reconstructed populations. These findings provide new insight into the relationships among different Crocus genotypes and show that AFLP analysis can be useful for Crocus diversity studies.

Phylogeny, geographic distribution, and new taxonomic circumscription of the Crocus reticulatus species group (Iridaceae)

Recent phylogenetic analyses proved several infrageneric units within the genus Crocus to be para- or polyphyletic. In an attempt to arrive at a system of Crocus that closely reflects species relationships, we provide here phylogenetic, morphometric, geographic, and nomenclatorial data for the species of a narrower-defined, monophyletic Crocus series Reticulati. We sequenced the ETS and ITS regions of the nuclear ribosomal DNA in 9 Reticulati and 19 outgroup species. Three chloroplast loci (trnL-F, rps16-trnQ, matK-trnK) were sequenced in the newly defined series Reticulati species and 1 outgroup. Data were analyzed with Bayesian and parsimony algorithms. The phylogenies resulted in 2 clearly separated, geographically defined species groups within the series Reticulati. The southern one comprises only the taxa from Turkey, while the species of the second group are distributed from Italy in the west through the areas north of the Black Sea to the Caucasus in the east. To arrive at monophyletic species we describe here C. danubensis sp. nov., C. filis-maculatis sp. nov., and C. orphei sp. nov. as new species, and we define C. reticulatus s.s. to comprise only the populations in the area north and east of the Black Sea.

Seed dormancy and germination of the subalpine geophyte Crocus alatavicus (Iridaceae)

Crocus alatavicus Regel et Sem. is a cormous perennial primarily distributed in central Asia that may have potential in horticulture; however, relatively little is known about seed dormancy in the genus Crocus. The primary aim of the present study was to identify the dormancy breaking and germination requirements of seeds of C. alatvicus and to assign them to a dormancy category. In its natural habitat, the underdeveloped embryo in C. alatavicus seeds grows in early summer, and radicles emerge in early autumn. However, cotyledon emergence is delayed until the following spring. Radicle emergence was promoted by warm stratification and cotyledon emergence by cold stratification. GA3 was ineffective in promoting either radicle or epicotyl emergence. We conclude that seeds of C. alatavicus have deep simple epicotyl morphophysiological dormancy of the type C1bB(root) – C3(shoot). To our knowledge, this is the first detailed study on the ecophysiology of seed dormancy and germination in the genus Crocus.

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.