*2.3. Melissopalynological Analysis*

### 2.3.1. Quantitative Pollen Analysis

Ten grams of honey were weighed and dissolved in 20 mL of distilled water. The solution was centrifuged for 10 min at 4500 rpm, and the supernatant was removed until a final volume of 5 mL. The sediment was stirred, and then two aliquots of 10 μL were deposited over a slide to microscopically examine (Nikon UK Ltd., Surbiton, UK). All pollen in the aliquot was counted. The results are expressed as the number of pollen grains per gram of honey considering the average value of the two aliquots.

### 2.3.2. Qualitative Pollen Analysis

Ten grams of honey were weighed and fully dissolved in 20 mL of distilled water. The solution was centrifuged for 10 min at 4500 rpm, and the supernatant was drawn <sup>o</sup>ff. The sediment was again washed with distilled water, and another centrifugation in the same conditions was done. Then, two aliquots (100 μL) of the sediment were deposited into a slide over a heated surface until desiccation. Lastly, drops were covered with a 24 × 24 mm cover glass containing a drop of glicerogelatin with fuchsine.

Slide observation was carried out with a light microscope at 400× or 1000×, as appropriate to improve the identification of pollen types. For each honey sample, at least 500 pollen grains were counted. All pollen grains were classified as the level of pollen type due to difficulties in accessing information about the wild botanical species of apicultural interest in a large part of the territory.

The relative frequency, expressed as a percentage, of all identified pollen type was considered for the pollen spectra of the samples. The following frequency classes were used to establish the representation of pollen types: dominant pollen (≥45%), accompanying pollen (from ≥15% to <45%), important pollen (from ≥3% to <15%), minor pollen (from ≥1% to <3%) and present pollen (≤1%). However, for unifloral honeys, the percentage of the main pollen type was calculated excluding plants considered to be non-nectariferous species, namely, *Acacia*, *Buxus sempervirens*, *Casuarina*, *Cannabis*, *Chamaerops*, *Cistus*, *Cyperus*, *Olea europaea*, *Papaver rhoeas*, *Paronychia argentea*, *Pinus*, *Pistacia*, *Poaceae*, and *Quercus*.
