Crocus sativus and its allies (Iridaceae)

Crocus sativus is an autumn-flowering species, unknown as a wild plant but long-cultivated for its scarlet style branches which yield Saffron, the dye and flavouring agent. There are several naturally-occurring related species from southern Europe and south western Asia which form a natural group within the genus.C. niveus from Greece is similar to these morphologically and is included here but is less closely related. The characters of the group are defined, a key to the taxa is provided and their relationships discussed.     

Pollen structure and germination of Crocus thomasii Ten. (Iridaceae)

Abstract

Mature pollen of C. thomasii Ten. (Iridaceae) has been studied from a morphological and physiological point of view and compared to that of C. sativus L., C. thomasii pollen is roundish, with a 83 μm diameter and a 14% of anomalous grains. Percentage of the positive alcoholdehydrogenase (ADH +) pollen grains is about 90% whereas the in vitro germinated rate reaches 55%. Exine is homogeneously thickened (2.5 μm) with randomly distributed thinner zones and 5 μm thick intine. Cytoplasm of vegetative and generative cells is very rich of small and large smooth vesicles and vesicles coated by ribosomes. The generative cell shows a thin ondulated pectocellulosic wall. Its nucleus is intensely fluorescent after treatment with the 4,6-diamidino-2-phenylindole (DAPI) fluorochrome. Although many ultrastructural aspects of the C. thomasii pollen are common in C. sativus. L., C. thomasii pollen is smaller, more regularly structured and germinates in vivo and in vitro in higher percentage than that of C. sativus.     

AFLP and MS-AFLP Analysis of the Variation within Saffron Crocus (Crocus sativus L.) Germplasm

The presence and extent of genetic variation in saffron crocus are still debated, as testified by several contradictory articles providing contrasting results about the monomorphism or less of the species. Remarkably, phenotypic variations have been frequently observed in the field, such variations are usually unstable and can change from one growing season to another. Considering that gene expression can be influenced both by genetic and epigenetic changes, epigenetics could be a plausible cause of the alternative phenotypes. In order to obtain new insights into this issue, we carried out a molecular marker analysis of 112 accessions from the World Saffron and Crocus Collection. The accessions were grown for at least three years in the same open field conditions. The same samples were analysed using Amplified Fragment Length Polymorphism (AFLP) and Methyl Sensitive AFLP in order to search for variation at the genetic (DNA sequence) and epigenetic (cytosine methylation) level. While the genetic variability was low (4.23% polymorphic peaks and twelve (12) effective different genotypes), the methyl sensitive analysis showed the presence of high epigenetic variability (33.57% polymorphic peaks and twenty eight (28) different effective epigenotypes). The pattern obtained by Factorial Correspondence Analysis of AFLP and, in particular, of MS-AFLP data was consistent with the geographical provenance of the accessions. Very interestingly, by focusing on Spanish accessions, it was observed that the distribution of the accessions in the Factorial Correspondence Analysis is not random but tends to reflect the geographical origin. Two clearly defined clusters grouping accessions from the West (Toledo and Ciudad Real) and accessions from the East (Cuenca and Teruel) were clearly recognised.     

Inter- and intraspecific genetic variation inHippophae (Elaeagnaceae) investigated by RAPD markers

Genetic diversity has been investigated by the application of molecular markers in, for the first time, all the taxa recognised in recent treatises of the genusHippophae. RAPD (random amplified polymorphic DNA) analyses were conducted with 9 decamer primers, which together yielded 219 polymorphic markers. We found 16 fixed RAPD markers, i.e. markers that either occurred in all plants of a population or were absent from all plants. Several of these markers were useful for analysis of interspecific relationships, whereas others can be considered as taxon-specific markers. Clustering of taxa and populations in our neighbour-joining based dendrogram was in good agreement with some recently suggested taxonomic treatises ofHippophae. Amount and distribution of genetic variability varied considerably between species. Partitioning of molecular variance withinH. rhamnoides supported earlier findings that a considerable part of the total variance resides among subspecies (59.6%) Within-population variability also differed considerably. Percentage polymorphic RAPD loci and Lynch and Milligan within-population gene diversity estimates showed relatively high values for some species close to the geographic centre of origin in Central Asia, e.g.H. tibetana and the putatively hybridogenousH. goniocarpa. Spatial autocorrelation analyses performed on 12 populations ofH. rhamnoides revealed positive autocorrelation of allele frequencies when geographic distances ranged from 0 to 700 km, and no or negative autocorrelation at higher distances. At distances between 700 and 1900 km, we observed deviations from the expected values with strongly negative autocorrelation of allele frequencies. A corresponding relationship between geographic and genetic distances could not be found when the analysis instead was based on one population from each of 8 species.     

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)     

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.     

Crocus antalyensis subsp. striatus subsp. nov. (Iridaceae) from southwest Anatolia

A new subspecies Crocus antalyensis subsp. striatus subsp. nov. from western Turkey is described. It is characterized by rough papery, light brown corm tunics, leaves that exceed the flower at anthesis and are recurved, distinctly striped inner perianth segments and deeply branched pale yellow or white style. The karyotype of the new subspecies is presented.     

Spatial and genetic structure within populations of wild American ginseng (Panax quinquefolius L., Araliaceae)

Spatial structure and fine-scale genetic structure were analyzed for the medicinal plant American ginseng (Panax quinquefolius L.) to more fully understand biological processes within wild populations. P. quinquefolius has been harvested for more than 250 years and is now considered threatened or rare throughout its range. Plants within four protected and four unprotected populations were significantly clumped based on Ripley's univariate analysis. Analysis with Ripley's bivariate test determined that juvenile plants were significantly clumped with adult plants at the shortest distance classes in all populations. Although plants were highly clumped, we found that significant fine-scale genetic structure was restricted to the shortest distance classes based on estimates of coancestry (f(ij)). In most cases, estimates of f(ij) were more significant among juveniles than among adults, especially at the shortest distance classes. The spatial structure of ginseng seems to result from the establishment and persistence of plants in favorable microhabitats coupled with limited seed dispersal around maternal individuals. There were no differences in patterns of fine-scale genetic structure between protected and unprotected populations.     

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.     

The genomic organization and evolutionary distribution of a tandemly repeated DNA sequence family in the genus Crocus (Iridaceae)

From a recombinant DNA-library from Crocus vernus, two closely related clones of highly repetitive DNA, pCvKB7 and pCvKB8, were sequenced and their genomic distribution and organization were investigated by Southern and in situ hybridization. The lengths of the clones were 181 and 178 bp respectively; the sequences were approximately 85% identical, and thus belonged to a sequence family, named the pCvKB8-family. No homologous sequences were found in the databases (BLAST made may 2004). The presence of pCvKB8 in 52 Crocus species and six species from other genera were analyzed by Southern hybridization. The sequence family was essentially Crocus-specific. However, the distribution of hybridization signal across the genus showed poor agreement with the taxonomic structure of the Crocus genus, suggesting that the subdivision does not follow the phylogeny of this sequence family. The chromosomal distribution on three Crocus species was essentially identical: tandem organization close to all telomeres and most centromeres, with a few additional intercalary sites.     

Isolation and characterization of a first set of polymorphic microsatellite markers in saffron, Crocus sativus (Iridaceae)

? Premise of the study: Twelve novel polymorphic microsatellite loci were developed and characterized from a repeat-enriched genomic library of Crocus sativus to study population and conservation genetics of this economically and medically important species. ? Methods and Results: The microsatellite loci were isolated using a modified Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) method. The average number of alleles per locus was 2.6. The observed and expected heterozygosities varied from 0.07 to 0.92 and 0.1 to 0.58, respectively. Polymorphic information content value ranged from 0.09 to 0.55 with an average of 0.34. Four out of twelve loci showed significant departures from Hardy-Weinberg equilibrium. ? Conclusions: The microsatellite markers reported here will be useful for evaluating genetic diversity and will likely serve as an ideal resource for use in marker-assisted breeding programs, germplasm analysis, and varietal identification.     

Molecular genetic diversity of musk deer Moschus moschiferus L., 1758 (Ruminantia, Artiodactyla) from the northern subspecies group

Genetic diversity within the northern subspecies group of musk deer Moschus moschiferus L., 1758 was examined based on the mtDNA control region hypervariable fragment (300-bp) sequence polymorphism. Nucleotide diversity, constituting 2.6% for the whole sample (n = 34), varied in the range from 0.6 to 1.9% for individual subspecies. Maximum values of this index were observed for Siberian subspecies (M. m. moschiferus), which had the widest range. Genetic similarity between the haplotypes of the musk deer from the Far East (Russia) and Sakhalin Island, which grouped in one cluster in a phylogenetic tree, was demonstrated. The data obtained indicate that the distribution of musk deer along the territory of Russia occurred from Eastern Siberia to the Far East, and from there to the Sakhalin Island. A currently observed decrease of the musk deer population number along with the increased habitat fragmentation can result in a decrease of the total genetic diversity and in inbreeding depression in the local isolated groups.     

Molecular genetic diversity of musk deer Moschus moschiferus L., 1758 (Ruminantia, Artiodactyla) from the northern subspecies group

Genetic diversity within the northern subspecies group of musk deer Moschus moschiferus L, 1758 was examined based on the mtDNA control region hypervariable fragment (300-bp) sequence polymorphism. Nucleotide diversity, constituting 2.6% for the whole sample (n = 34), varied in the range from 0.6 to 1.9% for individual subspecies. Maximum values of this index were observed for Siberian subspecies (M. m. moschiferus), which had the widest range. Genetic similarity between the haplotypes of the musk deer from the Far East (Russia) and Sakhalin Island, which grouped in one cluster in a phylogenetic tree, was demonstrated. The data obtained indicate that the distribution of musk deer along the territory of Russia occurred from Eastern Siberia to the Far East, and from there to the Sakhalin Island. A currently observed decrease of the musk deer population number along with the increased habitat fragmentation can result in a decrease of the total genetic diversity and in inbreeding depression in the local isolated groups.     

Inter- and intraspecific variation in the genitalia of the ‘Melitaea phoebe group’ (Lepidoptera, Nymphalidae)

The ecology and phylogeny of the Melitaeini butterflies have received considerable attention in the last few years. Several publications have been based on research using molecular methods. Genital morphometry has already been published on two sibling species but without any accompanying taxonomic revision of related species or their intraspecific taxonomy. In this study the morphology of genitalia was analysed by landmark-based geometric morphometrics in males and by traditional morphometrics in females. Based on these morphological characters, the whole M. phoebe group (subg. Cinclidia) was revised. According to our results M. telona Fruhstorfer, 1908 is conspecific with M. ‘phoebe’ ornata Christoph, 1893. Thus, we consider M. telona a junior synonym of M. ornata, and we propose new combinations: M. ornata ornata Christoph, 1893, M. ornata telona Fruhstorfer, 1908, M. ornata amanica Rebel, 1917, M. ornata capreola Varga, 1967, M. ornata emipunica Verity, 1919, M. ornata enoch Higgins, 1941, M. ornata kovacsi Varga, 1967, M. ornata nigrogygia Verity, 1938, M. ornata ogygia Fruhstorfer, 1907 and M. ornata totila Stauder, 1914 based on the rule of priority. The validity of the species status of M. punica and M. scotosia was confirmed, whereas M. sibina and M. pseudosibina could not be separated from M. phoebe using morphometrics. A new species from Iran (Zagros Mts.) was described as M. zagrosi sp. n. In addition, the whole geographical range of M. ornata was reconsidered and some biogeographical connections were discussed. In M. ornata four morphotypes were found based on genitalia: 1. ‘Western type’: including the Italian, Hungarian and SW Ukrainian populations; 2. ‘Eastern type’: with a much extended distribution ranging from the Balkan Peninsula through Asia Minor to Kazakhstan; 3. ‘The Levant type’ (=M. ornata telona comb. nov.); 4. ‘Northern Iranian type’ (Kopeth Dagh and Elburz Mts.). The distribution of these morphotypes can be attributed to historic separation in distinct glacial refugia.