The pollen consists of a resistance structure whose function is to protect the sperm nuclei of the plant; its external wall, called exine, is made of the natural polymer sporopollenin, which reacts to some environmental factors such as temperature and pH, allowing the generative cells to invade the stigma and fertilize the ovules of the flower [1].

This causes these same environmental factors to destroy the generative cells of the grains that do not reach the flower, but keeping the exine intact, which is why Erdtman [2] defines palynology as the study of this structure, considering its shape, ornaments, and thickness. As a fingerprint of each plant species and proposes acetolysis, a cleaning method that has been widely used by palynologists around the world as a classic working method.

However, the grains of some plant species are seriously damaged by this and other traditional chemical processes used to remove the protein cover of the exine and eliminate the generative cells, such as some species of the Zingiberales group, within which the genus Heliconia (Heliconiaceae) has grains that are easily destroyed by the acetolysis method [3][4].

Other families show slight damage to the exine, such as the species of Droseraceae [5], Araceae [6], Bromeliaceae, Cactaceae, and some whose grains have dispersion units of the tetrad and polyad type, such as Annonaceae [7], Fabaceae [8], Mimosaceae [9] and some genera of Rubiaceae [10] and Cucurbitacea [11].

This is a problem when carrying out pollination ecology studies, since valuable information is lost when processing samples, as well as it is important to have a laboratory and a series of reagents and equipment with high economic value that not all researchers or institutions have available provision.

This work aims to propose a fresh pollen fixation technique based on a glycerinated gelatin recipe that can be easily used in the field, and whose final preparations are suitable for long-term conservation in biological collections.

In total, 23 successful samples were prepared in fixed slides (Table 1), 20

Table 1. Presence of pollen in slides.

corresponding to pollen grains and 3 to Lycopodiaceae spores (Figure 1 and Figure 2). Four of the samples collected at the first site corresponding to the species Argeratina anisochroma (Asteraceae), Prunella vulgaris (Lamiaceae), Hydrocotyle ribifolia (Apiaceae) and Stereocaulon ramulosum (Stereocaulaceae) did not present pollen grains, and only the first could be collected again on Site 2 and get a workable mount to work with. Finally, the species Diplostephiumschultzii (Asteraceae) was only found in Site 2, however, it was not possible to mount a viable sample, because the flowers found were already dry and did not contain pollen.

Figure 1.Species collected at Site 1. Vibrum costarricanum (a), Diplostemum costarricense(b), Gnaphalium roseum (c), Senecio orstedianum (d), Valeriana prinophylla (e), Trifolium repens (f), Valeriana prinophylla (g), Sigesbeckia agrestis (h), Cirsium subcoriaceum (i), Acaena cylindrisachya (j), Solanum storkii (k).

Figure 2.Species collected at Site 2. Castilleja irazuensis (a), Hyepricum costarricense (b), Argeratina anisochroma (c), Chaetolepis cufodontisii (d), Lycopium thyoides (e), Lycopodium cotigum (f), Huperzia talamancana (g), Arcytophyllum lavarum (h), Bomareacostarricensis (i), Chusquea subtessellata (j), Gaultheria gracilis (k).

After the test time to verify the growth of fungi in the slides, no growth of the same was found in any of the samples, nor alteration in the structure of the pollen grains.

The method for dispensing fuchsin in the field with syringes greatly facilitated the work, avoiding the loss of pollen that can sometimes occur when handling or storing some flowers in bags. On the other hand, the grains are perfectly clean and viable for photography, without the need to apply Erdtman Acetolysis [2], which saves many economic resources, mainly in terms of equipment and reagents, and avoids the need to mount a lab, or access one.

However, it is necessary to generate specific pollen collections by sites or regions, since many families have very similar morphologies that can create confusion, especially within the Asteraceae family.

On the other hand, although Xylol gave very good results in preventing pollen growth from 2018 to the present, in tests subsequent to the execution of this methodology, it was replaced with Herb.io^® brand oregano essential oil because it contains Thimol, and in the same way as with Xylol, there was no fungal growth, so both reagents are suitable as a preservative for glycerinated gelatin.

We concluded that the field mounting method is completely feasible for rapid pollination ecology studies, providing the following advantages for researchers:

1) Samples with clear grains visible under an optical microscope.

2) Shorter sample processing time.

3) Conservation of samples in scientific collections without generating fungi even in natural environmental conditions.

To the National Museum of Costa Rica for the opportunity to collect the samples during the characterization tours of the Costa Rican páramo, as well as for hosting the witness samples. To Mr. Jorge Tello from the Dr. Bee beekeeping center (Bogotá, Colombia) for the training that gave rise to this assembly technique. And finally, to Joaquín Sánchez and Loengrin Umaña for the identification of the paramo plants.