MORPHOLOGICAL CHARACTERIZATION, MOLECULAR ANALYSIS AND CHROMOSOME COUNTING FOR THE IDENTIFICATION OF SERAPIAS HYBRIDS (ORCHIDACEAE)

The regional natural Park of Portofino hosts a conspicuous number of about 30 orchid species, among which Serapias lingua L., S. neglecta De Not., and S. parviflora Parl. are widely recognized within the area, while S. vomeracea Burm.f. and S. cordigera L. are only cited in literature but have not been recorded recently. Spontaneous hybrids, such as Serapias ×meridionalis E.G.Camus (S. lingua × S. neglecta), have been reported, but unclearly defined units were also observed ("S. olbia type"). Therefore, a morphological and molecular investigation, as well as chromosome counting, were carried out on different accessions, species and hybrids, currently present in the Park, aimed to characterize the existing entities and clarify their classification. Such morphological markers as hairiness of hypochile and epichile, type of callosity, length of labellum, width of epichile, width of hypochile and position of epichile were considered. Genomic DNA was extracted from leaves and molecular analyses were performed using Tubulin-Based Polymorphism (TBP) and Start Codon Targeted (SCoT) marker systems (1-2). Data were collected and analyzed by NTSYSpc 2.0 software with different coefficients, and estimated similarity values were used for cluster analysis. Dendrograms revealed, for both morphological and molecular analyses, a strict correlation among S. ×meridionalis and the "S. olbia type" unit, with values greater than those observed for parent species. This suggests that the accession defined as "S. olbia type" could be referred to S. ×meridionalis, while morphological differences (plant height or hairiness degree) could be related to the role of parents in the cross (e.g. S. lingua as ovule donor and S. neglecta as pollen donor, or vice-versa). A comparison with typical S. olbia is needed to verify this hypothesis. Chromosome counting was performed on root apices from S. ×meridionalis and its parental species. A combined protocol, specific for the Italian endemic Serapias species, was developed (3, 4), which allowed us to confirm the chromosome number of S. neglecta as having 2n = 36 chromosomes, and to characterize S. ×meridionalis as as 2n = 54. These findings have provided evidences for a correct identification of orchid hybrids, upholding the existence in the Park of spontaneous crossings between diploid (S. neglecta) and tetraploid (S. lingua, 2n =72) species.