Nigritella widderi (Orchidaceae) from central Apennines (Italy): evidence of plastome differentiation events

Abstract The present study focuses on the taxonomic and systematic classification of the tetraploid species Nigritella widderi , a rare orchid distributed in the eastern Alps (i.e., Austria, Germany, Italy, Slovenia) and in central Apennines (Italy). The research was fueled by the hypothesis that geographic isolation—or alternatively, long-distance seed dispersal—is driving the genetic differentiation of the Apennine population from its original Alpine lineage. Thus, a representative series of Apennine samples were subjected to genetic analyses, that is, characterization of 5 plastid Variable Number Tandem Repeats (VNTRs) (specifically loci 1, 2, 10b, 11b, and 19) and a nuclear barcode region (i.e., Internal Transcribed Spacer of rDNA, ITS). The plastid markers proved to be the key, providing clear elements of distinction at loci 10b and 11b between the Apennine and Alpine series. Interestingly, a potential novel Single Nucleotide Polymorphism (SNP) at locus 10b able to discriminate specifically N. widderi from the Apennines was identified. Conversely, the ITS barcode region showed insufficient discriminatory power for inter- and intra-specific distinction, perhaps due to its low mutation rate or the limited comparative dataset in the literature. In conclusion, this contribution represents the first molecular evidence that the Apennine population of N. widderi is genetically distinct, at least at the plastid level, from its Alpine counterpart. These findings are consistent with active microevolutionary processes shaped by unique environmental pressures, opening new questions about the biogeographical history of this taxon.