Thank you.

Hi José. Usually polytomies indicate lack of resolution in your data.  A hypothesis of hybrid origin (introgression) cannot be rulled out, but the short terminal branches may also indicate weak support for a hypothesis of introgression. Why do you think that your particular tree indicate a hybrid origin of the ITS2? With a single marker it is easy to manually verify the origin of each mutation. For each polytomy select three specimens representing the suspected parent (monoclonal) populations, and the suspected hybrid, and visually compare their aligned sequences. Make a table classifying the various types of nucleotide similarities: positions with nucleotides unique to a each specimen, and positions with pairs of nuceotides shared by each of the three possible pairs of sequences. For ambiguous base calls (presumed polymorphic positions) consider the individual nucleotides comprising the ambigous call (but check the chromatograms to make sure that the polymorphisms are real and not the result of poor quality chromatograms).  If the frequency of pairs of shared nucleotides are large and close to 50%, 50%, 0%, the hypothesis of hybrid origin is preferred; otherwise alternative hypotheses, such as lack of ancestral resolution or stable polymorphism may be preferred.