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.

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.

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.