Seed dormancy and germination in three Crocus ser. Verni species (Iridaceae): implications for evolution of dormancy within the genus

The aim of this work was to examine whether seed ecophysiological traits in three closely related Crocus species were associated with ecological niche differentiation and species divergence. Seeds of the temperate tetraploid cytotype of Crocus neapolitanus, the sub‐Mediterranean C. etruscus and the Mediterranean C. ilvensis were placed either on agar in the laboratory under different periods of simulated seasonal conditions or in nylon mesh bags buried outdoors to examine embryo growth, radicle and shoot emergence. In agreement with the phenology observed outdoors, in the laboratory embryos required a cool temperature (ca. 10 °C) to grow to full size (embryo length:seed length, E:S ratio ca. 0.75) but only after seeds received a warm stratification; radicle emergence then followed immediately (November). Shoot emergence is a temporally separated phase (March) that was promoted by cold stratification in C. neapolitanus while in the other two species this time lag was attributed to a slow continuous developmental process. These species have similar embryo growth and radicle phenology but differ in their degree of epicotyl dormancy, which is related to the length of local winter. Conclusions from laboratory experiments that only consider root emergence could be misleading; evaluating the phenology of both root and shoot emergence should be considered in order to demonstrate ecologically meaningful differences in germination behaviour and to develop effective propagation protocols. Although these taxa resulted from recent speciation processes, the outcomes suggest an early onset of adaptation to local ecological factors and that phylogeny may represent a significant constraint in the evolution and expression of seed traits in Crocus.     

Chromosome numbers of Turkish Crocus (Liliiflorae,Iridaceae) and their geographical distribution

The genus Crocus is known for its widely varying chromosome numbers (from 2n = 6 to 2n = 70) with varying numbers occurring even within species, as it is the case for Crocus biflorus Miller (2n = 8, 10, 12, 14, 16, 18, 20, 22, 24). After we found morphological diverse C. biflorus populations in Turkey doubts arose about their rank of being subspecies of the Italian C. biflorus (2n = 8). Here we publish the chromosome numbers for 76 populations of C. biflorus sensu lato distributed all over Turkey. The chromosome numbers ranged from 2n = 8 to 2n = 36, with the higher numbers occurring in the mountain ranges of the Anatolian Diagonal and east of it, while lower numbers were found only southwest of these mountains. Closely related taxa with similar distribution mostly differ in their chromosome numbers. This led us to assume that chromosomal changes influence speciation processes in the genus. Therefore, chromosome numbers may represent an important character for the establishment of a new taxonomic treatment of the Crocus species, especially within section Nudiscapus. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Taxonomy of the Poa laxa group,including two new taxa from Arctic Canada and Greenland,and Oregon,and a re‐examination of P. sect. Oreinos (Poaceae)

The Poa laxa Haenke species group, comprising four alpine–arctic taxa in P. sect. Oreinos, has a complicated and confusing taxonomic history. Here we re‐examine the taxonomy of the group and section based on sequences of three plastid and two nuclear ribosomal DNA markers. Poa laxa s.l. resolved in a clade with species of sections Abbreviatae, Oreinos, Stenopoa, and Tichopoa. In the plastid analyses, Poa laxa s.s. (type of sect. Oreinos) was placed in a subclade with P. glauca and other sect. Stenopoa species, while all other P. laxa s.l. taxa were placed in a subclade with species of sect. Abbreviatae. We maintain P. laxa s.s. (mainly 2n = 28) of west–central Europe in sect. Oreinos, while the other P. laxa s.l. taxa from northern Europe and North America (all 2n = 42 or higher ploid) are referred to P. sect. Abbreviatae. In North America only one collection of the northern European P. flexuosa subsp. flexuosa is accepted (Greenland, Jensen's Nunatakker), and P. flexuosa subsp. fernaldiana is accepted based on populations in eastern Canada and northeast USA. A new subspecies, P. flexuosa subsp. consauliae, is described from eastern Arctic Canada and Greenland; its variable morphological characteristics suggest introgression with P. glauca and it is predicted to be apomictic. Rocky Mountain P. laxa subsp. banffiana is treated as P. banffiana. Oregon material formerly considered to belong to P. laxa s.l. is named P. wallowensis. Poa sect. Oreinos is thus found to be polyphyletic, and is here emended to comprise only three continental European species, including P. laxa. Lectotypes are designated for P. aspera var. laxiuscula, P. fernaldiana, P. flexuosa, and P. laxa var. debilior. New chromosome numbers are reported for P. flexuosa subsp. consauliae (ca 2n = 42), and P. glauca (2n = 63) from Baffin Island. A key to the species and subspecies found in North America is given.     

Self-incompatibility inCrocus vernus subsp.vernus (Iridaceae)

Outcross, self- and mixed pollinations were performed inCrocus vernus subsp.vernus, a species with bicellular pollen, dry stigmas and hollow style. No differences were noted among the above pollinations concerning the germination of pollen and the growth of pollen tubes until the top of ovary. Within 45 min after pollinations 62% of pollen grains germinated. Pollen tubes penetrated the papilla cuticle extending along the papilla wall; on entry into stigmatic lobes they continued growth in the stylar secretion to ovarian locules. Here, however, self-pollen tubes failed to reach or to enter the ovule micropyle; while pollen tubes from either outcross- or mixed pollinations grew until fertilizing ovules. These observations gave evidence of a self-incompatibility system inCrocus, which appeared to be neutralized by mentor effect. The ovary as site of incompatibility response is discussed.     

The avoidance of self-interference in the Tuscan endemic spring geophyte Crocus etruscus Parl. (Iridaceae)

Spatial separation between sexes within hermaphrodite flowers (herkogamy) is widespread among angiosperms and is traditionally viewed as an adaptation that reduces the likelihood of self-pollination. While different degrees of herkogamy have been reported for Crocus, the relationship between sexual organ positions and reproductive fitness has not been explored yet, and studies of the breeding system within natural populations remain scarce within the genus. We analyzed the effects of different breeding systems (spontaneous self-pollination, facilitated self-pollination and cross-pollination) and of herkogamy degree on female reproductive success of the Tuscan endemic Crocus etruscus. Results showed that C. etruscus is monomorphic for style length and self-incompatible (SCI = 0.29). We also detected a significant negative effect of herkogamy on seed set of open-pollinated flowers. Thus, we conclude that the species avoids self-fertilization by self-incompatibility, but interestingly shows also a certain degree of spatial separation of male and female reproductive structures within the same flower. This floral trait could be useful to avoid self-interference thus reducing ovule discounting and pollen loss on its own stigma.     

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

Background and Aims: 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.Methods: 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.Key Results: 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.Conclusions 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.     

Taxonomy and cytogeography of Cardamine raphanifolia and C. gallaecica (Brassicaceae) in the Iberian Peninsula

For a long time it was considered that Cardamine raphanifolia is widely distributed with several infraspecific taxa in the Mediterranean region. Recent studies showed that C. raphanifolia is restricted to the northern Iberian Peninsula. Cardamine gallaecica from NW Spain was regarded to be closely related, but was originally regarded as C. amara, and later described as C. raphanifolia subsp. gallaecica. In this study, we validate the treatment of C. raphanifolia and C. gallaecica as distinct species, and show their clear differentiation based on morphometric and molecular AFLP analyses. Moreover, both species are clearly distinct from C. amara subsp. pyrenaea, which has an overlapping distributional range with C. raphanifolia. Hexaploid (2n = 48) and octoploid cytotypes (2n = 64) of C. raphanifolia, and tetraploid (2n = 32) and hexaploid (2n = 48) cytotypes of C. gallaecica were found. Due to their weak morphological and genetic differentiation, these polyploid races are not classified as separate taxa.     

Variation in Saffron (Crocus sativus L.) accessions and Crocus wild species by RAPD analysis

Crocus, a genus of Iridaceae is mostly grown in areas with Mediterranean environment as the best region. Saffron (C. sativus) is a perennial plant and is cultivated as an industrial crop in several regions of Iran. In this research, five accessions of cultivated Saffron from five areas in Khorasan, and Esfahan including Gonabad, Ferdows, Torbat-e-Heyidariye, Estahbanat, Gopayegan were used. Other nine species of saffron were grown naturally in Iran; so we collected two wild species (C. caspius and C. speciosus) from north of Iran (Gilan Province). RAPD markers were used to classify these species and to find their relationships. In the results of this study, the cluster analysis showed two distinct groups. Also, the maximum similarity was seen between C. caspius and C. speciosus (0.82) and the minimum was between Estahbanat, Ferdows accessions and C. speciosus (0.33). Finally, this method as a convenience procedure could be used to separate different accessions and species of Crocus as well.     

RAPD Analysis in Crocus sativus L. Accessions and Related Crocus Species

In the present paper a Random Amplified Polymorphic DNA (RAPD) investigation was carried out on DNAs from five Crocus sativus L. (saffron) accessions cultivated in different countries and on six closely related Crocus species. Aims of the study are to check whether cultivated saffron has maintained a constant genomic organisation and to clarify its relationships with possible ancestor species. For the fifteen primers, which produced positive results, DNAs of saffron corms from different accessions present the same amplification pattern, in accordance with the similar DNA content and base composition pointed out in previous studies. The amplification of the seven Crocus species DNAs with twenty-one primers provided 217 repeatable and interpretable fragments, which were scored for presence/absence and employed for a cluster analysis. Results indicated that C. sativus is very closely related to C. cartwrightianus and also similar to C. thomasii. This result, concurring with part of the previous evidence, would rule out the hypothesis of close relationships between C. sativus and C. pallasii.     

Chromosome numbers, karyotypes and nuclear DNA contents from perennial polyploid groups ofCerastium (Caryophyllaceae)

Somatic chromosome numbers have been determined for the followingCerastium taxa:C. eriophorum (2n = 36),C. alpinum (2n = 72),C. transsylvanicum (2n = 108),C. arcticum (2n = 108),C. latifolium (2n = 36),C. carinthiacum (2n = 36),C. banaticum (2n = 36),C. arvense subsp.glandulosum (2n = 36),C. arvense subsp.arvense (2n = 72) andC. fontanum (2n = 144). Karyotypes of three diploid species (C. eriophorum, C. banaticum andC. latifolium), belonging to three different taxonomic groups, were analysed and found to be similar. The relative nuclear DNA contents of all taxa were determined by flow cytometry and, for five species, also by Feulgen cytophotometry. The values obtained by the two methods are similar. A comparison of nuclear DNA contents among diploids shows that values differ significantly between different taxonomic groups, and are correlated with average chromosome size. Within closely related polyploid groups nuclear DNA amounts increase from 2x- to 4x- and 6x taxa as 1 : 1.4 : 2.4 in theC. alpinum complex, whereas DNA amounts are doubled comparing 2x- and 4x-subspecies in theC. arvense complex.     

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.     

Centaurea rahiminejadii sp. nov. and a new record in the genus Centaurea (Asteraceae,Cardueae–Centaureinae) from Iran

A new species, Centaurea rahiminejadii Negaresh (Asteraceae) in C. sect. Cynaroides Boiss. ex Walp., is described and illustrated from the Kermanshah Province, west Iran. It is morphologically similar to Centaurea regia Boiss. subsp. regia. Diagnostic morphological of this and closely related taxa are discussed and presented. The ecology, habitat and conservation status of the new species are also described. In addition, Centaurea regia subsp. cynarocephala (Wagenitz) Wagenitz is reported as a new record to the flora of Iran. Finally, the geographic distribution of the new species and closely related species is presented and mapped.     

Allium purpureoviride sp. nov. (Alliaceae) from east Anatolia,Turkey

Allium purpureoviride M. Koyuncu & ?. Genç (sect. Melanocrommyum) is described as a new species from east Anatolia, Turkey. The new species is closely related to A. rothii Zucc. from Israel and some characters of the new taxon show similarity with A. cyrilli Ten. and A. asclepiadeum Bornm. The morphological characters of the related taxa are discussed and the distribution of the taxa is mapped. The diploid chromosome number is 2n = 16.     

Ultrastructure and Germination Percentage of Crocus biflorus Miller subsp. biflorus (Iridaceae) Pollen

Pollen of Crocus biflorus Miller subsp. biflorus from natural habitats of Tusculum (Frascati, near Rome, Italy) has been studied in order to compare its structure and physiology to pollen of other Crocus species belonging to the Crocus sativus group. Mature pollen grains are rounded, 60 μm in diameter, in-aperturate (but with surface incisions where exine is lacking). DAPI staining reveals a spindle-shaped generative nucleus which is intensely fluorescent, and vegetative nucleus which is less fluorescent, and is elongated with numerous lobes. At anthesis the pollen is bicellular, but about 2% of tricellular grains occur among the pollen grains released from the anthers as well as on both naturally or handpollinated stigmas. Pollen germination is low in vitro, but higher in vivo. The pollen tubes are of normal shape. An electron-dense surface coat is sometimes visible on the exine, which in many cases, is detached from the exine. The vegetative cytoplasm is very rich in glycolipid bodies surrounded by endoplasmic reticulum. The generative cell has a lobed cell wall and is surrounded by the vegetative nucleus.     

Jurinea cataonica subsp. mardinensis subsp. nov. (Asteraceae) from southeast Anatolia,Turkey

A new subspecies, Jurinea cataonica Boiss. & Hausskn. subsp. mardinensis B. Do?an & A. Duran (Asteraceae), is described and illustrated from Anatolia, Turkey. The subspecies grows on abandoned fields in the Bakirkiri place (C8 Mardin Province) in southeastern Anatolia. It is morphologically similar to J. cataonica subsp. cataonica. Diagnostic morphological characters between these closely similar taxa are discussed and arranged in a key. Ecology and conservation status of the subspecies is also presented. In addition, the geographical distributions of the new subspecies and other related species are given.     

Comparison of the Giemsa C-banded karyotypes of the three subspecies ofPsathyrostachys fragilis,subspp.villosus (2x),secaliformis (2x, 4x), andfragilis (2x) (Poaceae), with notes on chromosome pairing

The karyotypes of diploidP. fragilis subsp.villosus (2n = 2x = 14) and tetraploid subsp.secaliformis (2n = 4x = 28) were studied by Giemsa C- and N-banding, and AgNO₃ staining and compared with the karyotype of subsp.fragilis (2x). The complements of subsp.villosus and subsp.fragilis were similar, with 8 metacentric and 6 SAT-chromosomes, one metacentric and two submetacentric pairs, with small to minute, polymorphic, heterochromatic satellites. The complement of subsp.secaliformis on the whole agreed with a doubling of the complement of diploidP. fragilis, suggesting autopolyploidy. Only the presence of 12 nucleoli in interphases identified 6 SAT-chromosome pairs. In subsp.villosus one or two extra micronucleoli indicated a chromosome pair with very low nucleolusforming activity, bringing the number of SAT-chromosome pairs to 4. This number may be a characteristc ofPsathyrostachys. Besides very small, inconsistently observed bands, the C-banding pattern consisted of 0–3 small bands per chromosome at intercalary and terminal locations, and at NORs. The level of banding pattern polymorphism was low, but enough to indicate that the taxa are outbreeders. Similarities in chromosome morphology and C-banding patterns identified homology of all chromosomes of subsp.villosus, but for 12 pairs only in subsp.secaliformis. Between plants, reliable identification of homology and homoeology (subsp.secaliformis) was possible only for the SAT-chromosomes and the shortest metacentrics. Chromocentres were very small and the amount of constitutive heterochromatin was low. N-banding stained chromosomes uniformly. The basic karyotypes of theP. fragilis taxa were similar to those ofP. juncea, P. lanuginosa, andP. stoloniformis supporting a close relationship and the presence of a common genome, N. NORs had different nucleolus-forming activities. Meiotic analysis demonstrated a high level of bivalent pairing in the three taxa. A chromosomal rearrangement was suggested in subsp.villosus. The low multivalent frequency in subsp.secaliformis indicates the presence of a pairing regulation mechanism. The majority of chiasmata were interstitial. Pollen grain size discriminated between diploid and tetraploid taxa. The existence of a diploid cytotype of subsp.secaliformis is supported by pollen measurements of herbarium material.     

Variation patterns within the genusCarex L. sect.Digitatae (Fries) Christ in Bulgaria

Seventeen natural Bulgarian populations of the species of the genusCarex sect.Digitatae were studied morphologically and karyologically. Stepwise discriminant analysis was used as a multivariate test for the separation of groups of populations from different taxa and to find a subset of characters contributing most for this separation. The following taxa were found in Bulgaria:C. humilis Leysser var.humilis; C. humilis var.longifolia Stoeva etPopova, var. n.;C. digitata L.;C. ornithopoda Willd. subsp.bulgarica (Vel.) Stoeva etPopova, comb. n. Aneuploid series of some chromosome numbers were found inC. digitata — 2n=48, 50, 52, 54, 56 and inC. ornithopoda subsp.bulgarica?2n=52, 53, 54, 56.C. humilis has 2n=36 and a much more heterogeneous karyotype than that of the above taxa. The values of the Euclidean distance between the populations vary within similar limits in both speciesC. humilis andC. digitata; this is not in accordance with the karyological results. The within and between population morphological variations were compared in each species. The first dominates in overall variation, which is probably connected with the very large populations and their mosaic space structure.     

Crocus sativus L. genomics and different DNA barcode applications

Crocus sativus L. is a sterile species (3n) whose origin has not been yet clarified. A lot of morphological studies supported the theory that it would have been originated from the evolution, or the hybridization, of other Crocus exemplars, especially C. thomasii, C. hadriaticus and C. cartwrightianus. Crocus sativus stigmas are saffron raw source but, because of their high economic value, sometimes this spice is adulterated. By the application of the DNA barcode technique, we analyzed different Crocus species genomes and we partially clarified some aspects of the phylogeny of this genus: in particular, C. sativus possible genetic derivation was elucidated. Our results also showed that different C. sativus species might have evolved by independent events, probably due to several geographical pressures. We demonstrated that barcoding method, usually adopted for interspecific taxonomic identification, could be also applied to intraspecific and population studies. Finally, we proposed this molecular approach as scientific tool able to discriminate and certificate saffron authenticity.     

Taxonomic and morphological studies inHypecoum sect.Hypecoum (Papaveraceae)

Hypecoum sect.Hypecoum comprises 8 species, one with 2 additional subspecies, in the Mediterranean area and the Middle East. Five of these are recognized for the first time, viz.Hypecoum procumbens L. subsp.fragrantissimum Å. E. Dahl,H. procumbens L. subsp.atropunctatum Å. E. Dahl,H. trullatum Å. E. Dahl,H. angustilobum Å. E. Dahl andH. torulosum Å. E. Dahl. H. dimidiatum Delile andH. pseudograndiflorum Petr., which have generally been included inH. imberbe Sm., are reestablished as distinct species. Morphology and variation patterns are described and discussed with special reference to mating systems. Self-incompatibility is dominating butH. procumbens subsp.procumbens andH. torulosum are self-compatible. The broad variation ranges of the former taxon in traits presumably related to mating system (petal, anther and stigma sizes as well as pollenovule ratios) indicate varying rates of outcrossing. SEM micrographs are given of petal and stigma surfaces and of pollen grains. Chromosome counts are presented for seven taxa. All are diploid with 2n = 16.     

Genetic diversity of Crocus antalyensis B. Mathew (Iridaceae) and a new subspecies from southern Anatolia

Crocus antalyensis B. Mathew is a bulbous plant endemic to Turkey. It is morphologically variable within the western part of Anatolia. Amplified fragment length polymorphism (AFLP) marker system was used to detect genetic variation among the Crocus taxa. Twenty-two primer combinations were used to screen for polymorphism among the samples. Genetic variation ranged from 0.44 to 0.69. We demonstrated the efficiency of the AFLP marker system for discriminating between individual C. antalyensis specimens. A high level of genetic variation was present among C. antalyensis specimens collected from different locations in Turkey. We also observed that C. antalyensis subspp. are genetically distinct from their relative Crocus flavus Haw. subsp. dissectus Baytop & B. Mathew. A new subspecies of C. antalyensis B. Mathew from southern Turkey is described. It is characterized by striped outer perianth segments, waist-shaped flowers, and glabrous throat of the perianth. A composite image of the new subspecies is presented.